Vaccine compositions having improved stability and immunogenicity

ABSTRACT

Disclosed herein are nanoparticles suitable for use in vaccines. The nanoparticles present antigens from pathogens surrounded to and associated with a detergent core resulting in enhanced stability and good immunogenicity. Dosages, formulations, and methods for preparing the vaccines and nanoparticles are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/819,962, filed on Nov. 21, 2017, which is a Continuation of U.S.application Ser. No. 15/257,436, filed Sep. 6, 2016, now issued as U.S.Patent No. 10,426,829, which claims priority to U.S. ProvisionalApplication Ser. No. 62/213.947. filed Sep. 3, 2015, U.S. ProvisionalApplication Ser. No. 62/255,786, filed Nov. 16, 2015, U.S. ProvisionalApplication Ser. No. 62/309,216, filed Mar. 16, 2016, and U.S.Provisional Application Ser. No. 62/350,973, filed Jun. 16, 2016, eachof which is herein incorporated by reference in its entirety for allpurposes.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:NOVV_060_04US_SeqList_ST25.txt, date recorded: Oct. 16, 2017; file size:91 kilobytes).

TECHNICAL FIELD

The present disclosure is generally related to nanoparticles useful forstimulating immune responses. The nanoparticles provide antigens, forexample, glycoprotein antigens, associated with a detergent core and aretypically produced using recombinant approaches. The nanoparticles haveimproved stability and enhanced epitope presentation. The disclosurealso provides compositions containing the nanoparticles, methods forproducing them, and methods of stimulating immune responses.

BACKGROUND

Infectious diseases remain a problem throughout the world. Whileprogress has been made on developing vaccines against some pathogens,many remain a threat to human health. Most notoriously HIV, for which avaccine remains elusive. Attempts have been made to produce vaccines tocertain pathogens but have resulted in failure that caused additionalpathology. Other pathogens also remain a problem, including Ebola, whichsporadically arises as epidemics—particularly in Africa—and gives riseto loss of life and global economic impact. Influenza virus is yetanother virus for which existing vaccine provide some protection buttechnical challenges in producing the virus mean that seasonal influenzavaccines may provide inadequate protection.

Deploying an effective vaccine relies on a combination of achievements.The vaccine must stimulate an effective immune response that reducesinfection or disease by a sufficient amount to be beneficial. A vaccinemust also be sufficiently stable to be used in challenging environmentswhere refrigeration may not be available.

Therefore, there is continuing interest in producing vaccines againstviruses that present public health issues throughout the globe and thereremains an ongoing need to produce effective vaccines with goodstability.

SUMMARY OF THE INVENTION

The present disclosure provides nanoparticles suitable for inducingimmune responses against pathogens. The nanoparticles offer improvedstability, as well as effective immunogenicity. In particular aspects,the pathogen is a virus and, typically, the antigen used to produce aviral nanoparticle is a viral glycoprotein.

In one aspect, the disclosure provides nanoparticles containing viralproteins that have enhanced stability. In some embodiments, thedisclosure comprises a vaccine composition comprising a nanoparticlecomprising a nonionic detergent, a viral glycoprotein, and apharmaceutical buffer. In typical embodiments, the nonionic detergentmay be selected from the group consisting of PS20, PS40, PS60, PS65, andPS80. In some embodiments, the composition does not comprise any freenonionic detergent. One or more glycoprotein antigen molecules surrounda detergent core, which contains the nonionic detergent, and thisprovides a nanoparticle structure that promotes immunogenicity andinhibits degradation of the antigen.

In some embodiments, antigen is selected from the group consisting of anRSV F protein, an influenza HA protein, an influenza NA protein, andcombinations thereof. Other antigens may be used, including Ebola.Typically, the antigen is a glycoprotein.

Optionally, the RSV F protein is a trimeric RSV F protein. The RSV Fprotein induces the production of neutralizing antibodies. In furtherembodiments, the neutralizing antibodies recognize the RSV F protein ina post-fusion state and/or a pre-fusion state. In a further aspect, eachPS80 particle may comprise between 4 and 7 RSV F proteins.

In some embodiments, an RSV F composition may comprise sodium phosphateat a concentration of between 15 mM and 25 mM; NaCl at a concentrationof between 125 mM and 175 mM; histidine between 0.25% and 2% w/v; andthe composition pH is between 5.8 and 7.2.

In some embodiments, an HA or NA influenza composition may comprisesodium phosphate at a concentration of between 15 mM and 25 mM; NaCl ata concentration of between 125 mM and 300 mM; histidine between 0.25%and 2% w/v; and the composition pH is above pH6.8 and typically belowabout pH 8.0.

In some embodiments, the composition comprises an adjuvant. In furtherembodiments, the adjuvant is alum or Martrix M™. In some embodiments,the composition does not comprise an adjuvant.

In some embodiments, a method of preventing infection comprisesadministering one or more doses of the vaccine composition. In someembodiments of the method, a single dose of the composition isadministered and induces a protective immune response. In someembodiments of the method, each dose consists of between about 100 μgand about 150 μg of the protein antigen. In further embodiments of themethod, the one or more doses are administered subcutaneously. In someembodiments of the method, the composition comprises an adjuvant. In afurther embodiment of the method, the adjuvant is alum. In someembodiments of the method, the composition is free of adjuvants.

In some embodiments of the method, one or more doses of the compositionare administered to an adult. In further embodiments of the method, theadult is a female, and the female may be pregnant. In furtherembodiments of the method, the adult is over the age of 65 or over 60.In some embodiments of the method, one or more doses of the compositionare administered to a child. In further embodiments of the method, thechild is a neonate or an infant.

For RSV vaccine, in some embodiments, a composition comprises aheterologous population of at least three RSV F nanoparticle types,wherein each nanoparticle comprises at least one RSV F protein trimersurrounding a detergent-containing core that comprises PS80, and whereinthe first RSV F nanoparticle type comprises anisotropic rods, whereinthe second RSV F nanoparticle type comprises spherical oligomers, andwherein the third RSV F nanoparticle type comprises intermediates ofanisotropic rods and spherical oligomers.

In some embodiments, a method of manufacturing an RSV F proteinnanoparticle comprises preparing an RSV F protein extract from a hostcell using a first detergent and exchanging the first detergent for asecond detergent, wherein the second detergent is PS80, and whereby thenanoparticle exhibits enhanced stability. In a further embodiment of themethod, the first detergent is NP-9. In some embodiments of the method,the enhanced stability is selected from protease resistance, oxidativestress resistance, thermal stress resistance, and resistance toagitation. In some embodiments of the method, the molar ratio of PS80:RSV F protein is about 35 to about 65.

In some embodiments, an RSV F nanoparticle comprises one or more RSV Fprotein trimers associated with a PS80 detergent core. The RSV Fnanoparticle, the nanoparticle has an average diameter of about 20 nm toabout 60 nm as measured by dynamic light scattering. In some embodimentsof the RSV F nanoparticle, each RSV F protein trimer contains an RSV Fprotein selected from the group consisting of RSV F proteins having adeletion of 1 to 10 amino acids corresponding to residues 137-146 of SEQID NO:2. In some embodiments of the RSV F nanoparticle, each RSV Fprotein trimer contains an RSV F protein selected from the groupconsisting of RSV F proteins having a deletion of 1 to 10 amino acidscorresponding to residues 137-146 of SEQ ID NO:2 and an inactivatedprimary fusion cleavage site.

In some embodiments of the RSV F nanoparticle, the RSV F proteincomprises a deletion of ten amino acids corresponding to residues137-146 or SEQ ID NO:2, and inactivation of the primary furin cleavagesite by mutation of arginine residues at positions 133, 135, and 136 toglutamine. In further embodiments of the RSV F nanoparticle, the RSV Fprotein comprises or consists of SEQ ID NO:19, which is the maturepeptide. In certain embodiments of the RSV F nanoparticle, the RSV Fprotein comprises or consists of SEQ ID NO:8. Vaccine formulationscontaining RSV F nanoparticles comprise substantially of the maturepeptide with some full-length peptide (SEQ ID NO:8). Over time, smallamount of truncated RSV F peptide may arise due to proteolysis.Advantageously, however, the RSV F nanoparticles disclosed hereinminimize such degradation and provide extended stability.

This application also discloses enhanced thermostability influenzananoparticles. Unlike prior influenza nanoparticles the methods andcompositions provided here exhibit resistance to trypsin and enhancedthermostability and thus immunogenicity.

For Ebola, the Ebola virus nanoparticles comprise an Ebola virusglycoprotein (GP) trimer attached to a non-ionic detergent core as wellas vaccine compositions containing the nanoparticles, optionally incombination with a Matrix M saponin adjuvant. In addition, thedisclosure provides for methods of inducing an immune response againstEbola virus in humans by administering a composition containing an Ebolavirus nanoparticle and a saponin adjuvant. Methods of protecting againstEbola infection are also provided.

Similarly, nanoparticles containing influenza proteins, either HA, NA orboth, are provided. HA nanoparticles showing trypsin-resistance, anindicator of proper folding are provided. Methods of protecting againstinfluenza infection using the influenza nanoparticles in vaccineformulations are also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B depict primary protein structures of RSV F proteins,accompanied by a polypeptide sequence. FIG. 1A depicts the primaryprotein structure of wild-type RSV A2 strain versus that of a modifiedRSV F protein. Furin cleavage sites are indicated by triangles. FIG. 1Bdepicts the amino acid sequence of a modified RSV F protein (SEQ IDNO:19); with the F1 domain in light-shaded text (residues 1-84), the F2domain in dark-shaded text (residues 85-539), black lines connectingcysteines that form disulfide bonds, underlined asparagines indicateN-linked glycosylation sites, light-shaded vertical dotted linesindicate a furin cleavage site, and dark-shaded vertical dotted linesindicate a major cleavage site.

FIG. 2 depicts the separation peaks of RSV F proteins by reverse phaseHPLC, wherein four major species are identified and correspond to the 4major peaks. The peak comprising the lowest molecular weight species˜51.2 kDa-˜51.3 kDa) is a soluble trimer; the next peak comprises a fulllength trimer (˜64.5 kDa) lacking fatty acids, and the final two majorpeaks are full length trimers wherein the trimers comprise palmitoleicacid (˜64.7 kDa) and palmitic acid (64.788 kDa), respectively.

FIG. 3 depicts the separation of RSV F proteins in a reducing SDS-PAGE.The largest molecular weight proteins comprise high molecular weightspecies, followed by variants comprising the F1 and F2 domains, thenjust the F1 domain and variants thereof, followed by just F2 domains.

FIG. 4 depicts a chromatogram output LC-UV peptide mapping that covers90% of the amino acids comprising the primary protein structure of theRSV F protein. The combined sequence coverage, including theearly-eluting peptides, was found to be 98%, confirming the amino acidsequence of the RSV F protein.

FIG. 5 depicts a glycoanalysis of a purified RSV F protein using FPLCcombined with fluorescence detection (FLD). The major glycan structuresdetected are fucosylated Man3 glycans.

FIG. 6 features an electron micrograph of RSV F nanoparticles with RSV Fprotein trimers associated with cores of PS80. The figure furtherdepicts a characterization of a single RSV F protein trimer featuringthe orientation of the F1 and F2 domains, antigenic site II which isrecognized by the Palivizumab antibody, and the C- and N-termini of theF1 domains further comprising fatty acids such as palmitic andpalmitoleic acids.

FIG. 7 depicts Dynamic Light Scattering (DLS) measurements of particlesize of RSV F nanoparticles. The DLS measurements show that the size ofthe nanoparticles is modulated by both the available PS80 and the RSV Fconcentration. An increase in PS80 at a fixed concentration of RSV Fconcentration results in a decrease in the average nanoparticle size(Z-ave).

FIG. 8 depicts the discrete molecular weight distributions of sampleconcentration versus molecular weight of the nanoparticles, wherein theconcentration of RSV F and the percentage of PS80 is varied. Thegreatest signal intensity of nanoparticles is achieved with 0.2% PS80and 1 mg/mL RSV F, suggesting greater uniformity of nanoparticles andconfirming the modulation of particle size as a combination ofconcentrations of PS80 and RSV.

FIGS. 9A and 9B depicts the shape of RSV nanoparticle types producedwith variable PS80 percentages and RSV F concentrations. FIG. 9A revealsthat a composition using 0.2% PS80 and 0.22 mg/mL RSV F produces threeprimary types, monomeric/dimeric anisotropic rods, spherical oligomers,and intermediates thereof. FIG. 9B reveals that a composition using0.05% PS80 and 0.22 mg/mL RSV results in a population dominated bymonomeric/dimeric anisotropic rods, whereas a composition using 0.05%PS80 and 1.0 mg/mL results in a population dominated by sphericaloligomers.

FIG. 10 depicts the effects of the stressors on particular subsectionsof the RSV F protein in a nanoparticle, as presented in a reduced Lys-Cpeptide map with relative abundance compared to a control. The stressorsare 50° C. for two weeks, pH 3.7 at 25° C. for one week, pH 10 at 25° C.for one week, oxidation of protein by hydrogen peroxide at 25° C. forone week, and agitation at 25° C. for one week.

FIG. 11 illustrates stability of the antigenic site 2 (palivizumab site)exposed to various stress conditions. The percentages are presented as arelative abundance compared to a control. The closer to 100% or over,the greater the resilience in light of the stress conditions. The dataillustrate the nanoparticles maintain excellent antigenic siteconsistency therefore yielding a stable immune response.NSELLSLINDMPITNDQK/K; SEQ ID NO:20 and LMSNN (SEQ ID NO:21) are portionsof antigenic site II.

FIGS. 12A, 12B, 12C, and 12D depict the stability of the RSV Fnanoparticle composition by showing murine immunogenicity after thenanoparticle compositions were exposed to environmental stress. The micewere sampled at day 21 for anti-RSV F IgG, day 35 for PCA titers, andday 35 for RSV/A neutralizing titers. FIG. 12A depicts the results forthe −70° C. control. FIG. 12B depicts the results a composition exposedto 50° C. for two weeks. FIG. 12C depicts the results for a compositionexposed to a pH of 10 at 25° C. for two weeks. FIG. 12D depicts theresults for a composition exposed to 0.5% hydrogen peroxide at 25° C.for one week.

FIG. 13 depicts the enhanced protease resistance of nanoparticles havinghigher PS80. Over a period of 18 months, RSV F nanoparticles formulatedin the presence of a higher PS80 percentage (0.03%) exhibited lessprotease degradation versus RSV F nanoparticles formulated in thepresence of lower PS80 percentage (0.015%), as evaluated by SDS-PAGE. Inaddition, fewer high molecular weight (HMV) structures were observedwith higher PS80 amounts.

FIG. 14 depicts the comparison of mAbs binding to RSV F nanoparticlesversus RSV F A strain viral protein, wherein the equilibriumdisassociation constant for site I, II, and IV antibodies reveal thatmAbs antibody binding at each of the sites is comparable.

FIG. 15 depicts the results of competitive binding assays in whichantibodies present in sera from cotton rats exposed to placeboconditions, RSV/A infection, formaldehyde inactivated RSV, RSV Fnanoparticles, and RSV F nanoparticles with alum were compared againstone another in binding to site I, II, and IV.

FIG. 16 illustrates a process flow chart for a method of makingnanoparticles disclosed herein.

FIG. 17 illustrates a flow chart for a method of making HA nanoparticlesdisclosed herein. Sf9 cells containing baculovirus-expressed HA aregrown and then the HA is extracted using the non-ionic detergent NP9.The extract undergoes sequential purification and a detergent exchangestep on Lectin affinity column, and is then filtered and formulated intoa bulk drug substance.

FIGS. 18A to 18F illustrate steps and results obtained using a method ofproducing influenza nanoparticles using the HA glycoproteins as anexample. FIG. 18A shows sequential purification steps from cellinfection, through cell lysis and three columns used to provide purifiednanoparticles (TMAE (trimethylaminoethyl) followed by lentil lectin,followed by a sulfate (SO3⁻) column). FIG. 18B illustrates achromatogram obtained using a TMAE column. FIG. 18C illustrates achromatogram obtained using a lentil lectin column purification step.FIG. 18D illustrates a chromatogram obtained using a sulfate columnpurification step. FIG. 18E shows a gel (upper right panel) of the TMAEand LL columns stages. The bottom panel shows a western blot of the gel.Lanes are as shown in the upper left panel. FIG. 18F shows a gel witheluate from the S03-column.

FIGS. 19A to 19J illustrate purity analyses of HA nanoparticles producedusing different sub-types and in different insect cell lines. FIG. 19Ashows a gel, western blot for HA, and gp64 for HA nanoparticlescontaining A/New Hampshire/1/2015 HA. FIG. 19B shows a quantification ofthe HA band, and shows that the HA is 99.1% pure by densitometry. FIG.19C shows a gel, western blot for HA, and gp64 for HA nanoparticlescontaining A/Switzerland/9715293/2013 HA. FIG. 19D shows aquantification of the HA band and shows that the HA is 94.5% pure bydensitometry. FIG. 19E shows a gel, western blot for HA and gp64 for HAnanoparticles containing A/Hong Kong/4801/2014 HA. FIG. 19F shows aquantification of the HA band and shows that the HA is 93.3% pure bydensitometry. FIG. 19G shows a gel, western blot for HA, and gp64 for HAnanoparticles containing B/Phuket/3073/2013 HA in Sf9 and Sf22a cells.The right hand panel shows a quantification of the HA band and showsthat the HA is 95.4% pure by densitometry. FIG. 19H shows a gel, westernblot for HA, and gp64 for HA nanoparticles containing B/Brisbane/60/2008HA in Sf9 cells. The right hand panel shows a quantification of the HAband and shows that the HA is 96.7% pure by densitometry. FIG. 19Imeasures HA purity using RP-HPLC. FIG. 19J summarizes the data for HAnanoparticles using three influenza A sub-types and two influenza Bsub-types.

FIG. 20 shows HA nanoparticles in electron micrographs.

FIGS. 21A and 21B shows a comparison of docking of HA trimers ontocryoEM structures for HA nanoparticles (FIG. 21A) and for the HA trimerson influenza VLPs containing both HA and NA proteins (FIG. 21B).

FIG. 22 illustrates a study with a combination nanoparticle compositioncontaining RSV F nanoparticles and a representative of HA nanoparticle.

FIGS. 23A to 23F illustrate results obtained according to the study inFIG. 22 . FIG. 23A shows HAI titer against the homologous strain. FIG.23B shows heterologous HAI titer against a heterologous strain. FIG. 23Cshows palivizumab competitive antibodies. FIG. 23D shows neutralizingantibodies against the RSV A strain. FIG. 23E shows T cell responsesagainst RSV F protein. The response obtained with Matrix-adjuvantednanoparticles is prominent. FIG. 23F shows T cell responses againstinfluenza protein.

FIGS. 24A-24C illustrate a process and results for obtaining HAnanoparticles that have enhanced stability. Notably, the pH range duringthis purification is in a neutral range of pH 7.0 to pH 7.4. FIG. 24Ashows purification steps from using thawed cells expressing the HAprotein through to the bulk drug substance (BDS) product. FIG. 24B showsa chromatogram trace from a representative nanoparticle using the A/NewHampshire 1/2015 strain. The flow-through from the column is collected,leaving undesirable products behind. FIG. 24C shows a chromatogram tracefor the detergent exchange step on a lentil lectin column. Theflow-through from this column is discarded as is the wash. Elution isperformed with 0.01% PS80. The buffers are as follows: A1: 25 mM sodiumphosphate, pH7.2, 150 mM NaCl, 0.01% PS80, A2: 25 mM sodium phosphate.pH7.2, 500 mM NaCl, 0.5% NP9, A3: 25 mM sodium phosphate, pH7.2, 150 mMNaCl, 0.1% PS80, B1: 25 mM sodium phosphate, pH7.2, 150 mM. The HAnanoparticles are then concentrated and stored in 0.05% PS80 buffer asshown in FIG. 24A.

FIGS. 25A-25D show results for purification of trypsin-resistantnanoparticles from several strains. FIG. 25A shows a representativestrain for an H1N1 subtype, A/New Hampshire/1/2015. FIG. 25B shows arepresentative strain for a B type influenza. R/Brisbane/60/08 HA. FIG.25C shows a representative strain for an H1N1 subtype, A/NewHampshire/1/2015. In each case the data shows high levels of productionand excellent purity. FIG. 25D provides a differential scanningcalorimetry (DSC) comparison of the trypsin resistant nanoparticlesversus nanoparticles produced using a process that exposes them to lowpH, about pH 6.0. The DSC, data shows greater thermostability with theneutral pH process establishing that the HA protein in the nanoparticleis properly folded.

FIGS. 26A-26C show results for enhanced trypsin resistance oftrypsin-resistant nanoparticles from several strains expressed in Sf9cells. Purified HA nanoparticles made in Sf9 insect cells are HA0. Whenexposed to trypsin HA0 is cleaved to HA1 and HA2 at Arg AA344 in H1.Correctly folded HA trimers will resist further cleavage when incubatedwith increasing concentrations of trypsin. FIG. 26A shows neutral pHpurified B/Brisbane/60/08 is resistant to trypsin thus is correctlyfolded (left panel) whereas acid pH purified B/Brisbane/60/08 HA1 istrypsin sensitive thus misfolded (right panel). FIG. 26B shows that acidpurified but not neutral-purified HA nanoparticles from A/HongKong/4801/2014 are mis-folded. FIG. 26C shows trypsin resistance ofneutral pH A/New Hampshire/1/2015 (H1N) HA nanoparticles. Correspondingacid pH purified nanoparticles were trypsin sensitive (not shown).

FIG. 27 shows trypsin sensitivity of a commercial egg-purified influenzavaccine (left panel) and a commercial recombinant influenza (rightpanel). HA0 is cleaved to HA1 and HA2 in the left panel. Properly foldedHA1 is resistant to further trypsin however. In contrast, the commercialrecombinant vaccine shows that the HA1 is degraded by trypsin,indicating mis-folded protein is present in the vaccine.

FIGS. 28A-28C shows induction of antibodies and protection frominfection. Mice were immunized SC on Days 0, 14, and 28 with 5 μgEBOV/Mak GP, 5 μg EBOV GP adjuvanted with 50 μg AlPO4 or 5 μg EBOV/MakGP adjuvanted with 5 μg Matrix-M. Serum was obtained on day 28 andevaluated by ELISA for anti-EBOV/Mak GP IgG (FIG. 28A) or anti-Ebolavirus neutralizing antibody (FIG. 28B). Black bars represent the groupGMT and error bars indicate 95% confidence intervals of the GMT. On day42, mice were infected with 1,000 pfu mouse adapted Zaire Ebola virusstrain 1976 Mayinga. Following challenge, mice were observed daily formorbidity and mortality for a period of 21 days. FIG. 28C showsKaplan-Meier survival curve for infected mice.

FIGS. 29A-29C show Matrix-M enhanced EBOV/Mak GP-specific IgG and IgGsubclass responses. Mice were immunized IM on Days 0 and 21 with 5 μg ofEBOV/Mak GP alone or combined with either 2.5 or 5 μg Matrix-M or 50 μgAlPO₄. Mice received PBS as placebo control. At days 21, 28 and 60following the first injection, serum samples were collected and testedfor EBOV/Mak GP-IgG (FIG. 29A), IgG1 (FIG. 29B) and IgG2a (FIG. 29C).The results are representative of two separate experiments. Black barsrepresent the group GMT and error bars indicate 95% confidence intervalsof the GMT.

FIGS. 30A-30D show Ebola nanoparticles with Matrix-M induced robust CD4⁺T cell and CD8⁺ T cell responses and multifunctional T cells. Spleencells were stimulated with Ebola/Mak GP peptide pools covering theentire GP sequence. Culture medium or PMA (50 ng/ml) plus ionomycin (200ng/ml) were used as negative and positive controls. IFN-γ positive spotsfrom day 28 (FIG. 30A) and 60 (FIG. 30B) were counted and analysed withan ELISPOT reader and associated software. Background numbers of themedium controls were subtracted from the numbers of peptides-stimulatedwells and a mean was derived from the triplicates. Cells from all fivemice in the same group at day 28 were pooled and incubated with eithermedium alone, or GP peptide pools, or PMA plus ionomycin for 6 hours at37° C. with the presence of BD Golgi-stop/Golgi-plug. Cells were thenharvested and stained for cell surface markers and intracellularcytokines. Frequency of cytokines was analysed using Flowjo software andFlowjo Boolean function by gating on live CD3+CD44+CD62−CD4+ effectormemory T cells or live CD3+CD44+CD62−CD8+ effector memory T cells.(FIGS. 30C and 30D) The value for single cytokines, double cytokines ortriple cytokines represent the sum of the frequency of cells expressingany one of the three cytokines (IFN-γ, TNFα and IL-2), any two of thethree cytokines or all three cytokines. The result is representative oftwo separate experiments. Black bars indicate group means and error barsrepresent standard deviation.

FIGS. 31A-31E show the Matrix-M enhanced Germinal Center (GC) cellresponse. Fresh splenocytes were stained for GC B cells and data wasacquired as described in Materials and Methods. Data was analysed withFlowjo software. Dead cells were excluded from analysis with InvitrogenLIVE/DEAD™ fixable yellow dye. (FIG. 31A), GC cells were defined asCD95⁺GL-7⁺ on B220⁺ B cell gate and the numbers in the dot-plot ofrepresentative mice indicate the mean and standard deviation of GCfrequency from all five mice in the same group at day 28. GC cellfrequencies from individual mice are shown for days 28 (FIG. 31B) andday 60 (FIG. 31C). The absolute GC cell number per spleen from days 28(FIG. 31D) and 60 (FIG. 31E) was calculated by multiplying the frequencyof GC cells within the total number of splenocytes in the spleen. Blackbars indicate group means and error bars represent standard deviation.

FIGS. 32A-32E: Matrix-M enhanced the frequency and absolute number ofT_(FH) cells in the spleen. T_(FH) cells, defined as CXCR5⁺PD-+1⁺ Tcells within B220⁻CD49b⁻CD3⁺CD4⁺ T cell gate, were identified in spleensat days 28 and day 60, Representative dot-plot of T_(FH) cell analysisfrom each group is shown (FIG. 32A). The number in the dot-plot is theaverage frequency and standard deviation from day 28. The frequency ofT_(FH) cells within the CD4+ T cell population from days 28 (FIG. 32B)and 60 (FIG. 32D) is shown. The absolute T_(FH) cell number per spleenfrom days 28 (FIG. 32C) and 60 (FIG. 32E) was calculated by multiplyingthe frequency of T_(FH) cells within the total number of splenocytes inthe spleen. Black bars indicate group means and error bars representstandard deviation.

FIGS. 33A-33B show Matrix-M induced long-lived plasma cells in bonemarrow. Spleen and bone marrow cells were incubated overnight inEBOV/Mak GP coated ELISPOT plates. The EBOV/Mak GP-specific IgG spotswere detected by incubating with goat-anti-mouse IgG-HRP followed byspot development. Spot numbers were counted and analyzed using anELISPOT reader. The number of antibody secreting cells (ASC) per millioncells is shown. (FIG. 33A) day 60 EBOV/Mak GP-IgG ASC number in thespleen; (FIG. 33B) day 60 EBOV/Mak GP-IgG ASC number in the bone marrow.Black bars indicate group means and error bars represent standarddeviation.

FIGS. 34A-34B show features of an Ebola Glycoprotein. FIG. 34A shows thedomain structure. FIG. 34B shows the amino acid sequence of a GP withthe cleaved signal peptide and the N- and C-terminii of the matureprotein, and the furin cleavage sequence (SEQ ID NO: 22).

FIGS. 35A-35C show electron micrographs of nanoparticles of thedisclosure. FIG. 35A illustrates a representative electron micrograph ofthe nanoparticles. Note that FIG. 35B illustrates the non-ionicdetergent core with from up to 5 copies of trimers attached to the core.In some cases, additional trimers are out of the plane of view. FIG. 35Cshows a docking study with GP trimers overlaid onto a nanoparticle froma micrograph.

FIG. 36 illustrates the ability of three monoclonal anti-Ebolaantibodies to detect the Ebola nanoparticles.

FIG. 37 shows the Surface plasmon resonance (SPR) data for binding ofthe antibodies to the epitopes of the Ebola. GP nanoparticles (SEQ IDNOs:23-25).

FIG. 38 illustrates the high potency of binding of the 13C6 antibody tonanoparticles of the disclosure.

FIG. 39 illustrates a Baboon immunogenicity study design. Group 1 was 60μg GP nanoparticles with no adjuvant. Group 2 was 60 μg GP nanoparticleswith 800 μg AlPO4 adjuvant. Group 3 was 60 μg GP nanoparticles with 50μg Matrix-M adjuvant. Group 4 was 5 μg GP nanoparticles with 50 μgMatrix-M adjuvant.

FIGS. 40A-40B illustrate results of the Baboon immunogenicity study inFIG. 39 . At Day 21, EC90 titers were increased for Groups 2 and 3. FIG.40A Titers were approximately the same in both groups and also againstnanoparticles containing glycoproteins from the Makona Ebola virus andthe Mayinga strain, which is the prototypical variant of the Ebola Zairestrain. As shown in FIG. 40B, by Day 31, the immune response waspronounces in all cases and especially for compositions containing GPand Matrix M adjuvant. Notably, the lower dose of GP (5 μg) performed aswell as the higher dose (60 μg) underscoring the dose-sparing effect ofthe Matrix-M.

FIG. 41 illustrates the durable immune response achieved by thenanoparticle compositions. The data shown in the EC50 GMT responses forIgG after administration at Day 0 and Day 21. The nanoparticles with GPand Matrix-M show better responses than an alum adjuvant and theresponses remain higher over time.

FIG. 42 illustrates the stimulation of the immune response involvingIFNγ releasing cells. The Matrix M combined with 5 μg GP nanoparticlesgave the maximum response followed by the higher dose GP nanoparticles(60 μg). Using alum provided a low but detectable increase in peripheralblood mononuclear cells (PBMC) secreting IFN-γ.

FIG. 43 illustrates the IFNγ and TNF-α, release profiles from CD4+ andCD8+ T-cells isolated from baboons that were administered vaccinecompositions containing the GP nanoparticles disclosed herein

FIG. 44 illustrates the cytokine release profiles from T-cells isolatedfrom baboons that were administered vaccine compositions containing theGP nanoparticles disclosed herein. The data show that MatrixM-adjuvanted GP nanoparticle compositions stimulate immune responseshaving broader cytokine release profiles.

FIG. 45 shows a vaccine trial design performed in Cynomolgus macaques.Animals were administered a vaccine composition of 5 μg GP+50 μgMatrix-M at Days 0 and 21 then challenged at Day 42. Animals 33360,33362, and 33355 were treated with the vaccine composition. Placebo wasadministered to animal 33356.

FIG. 46 shows the IgG titers obtained in the Cynomolgus macaquetrial. ByDay 28, EC50 titers had exceeded 10⁵.

FIGS. 47A-47C shows induction of IFN-γ secreting PBMC cells isolatedfrom treated macaques. Peptides derived from Ebola Zaire GP were pooledand used in the assay. A consensus peptide derived from the Zaire andSudan strains was also tested. The data shown illustrates cellsresponding to those peptides at Week 0 (FIG. 47A), Week 3 (FIG. 47B),and Week 5 (FIG. 47C). The control animal injected with placebo showedessentially no response. In contrast, vaccine-treated animals showed arobust increase in cells releasing IFN-γ in response to the variouspeptides tested.

FIG. 48 shows viral load and survival in macaques. By Day 7post-challenge the placebo animal exhibited a substantial increase inviral nucleic acid, indicating Ebola infection. By Day 9 the animal waseuthanized. All vaccinated animals survived. Only animal 33360 exhibiteda detectable increase in viral nucleic acid, which was about the limitof detection. By Day 10, even in that one animal, viral RNA levels haddropped beneath the ability of RT-PCR to detect them.

FIG. 49 shows a vaccine trial design for an additional macaque study.Animals were administered saline or 5 μg GP+50 μg Matrix-M. Group Freceived vaccine at weeks 0 and 6. Group G received vaccine at weeks 0and 3. Both groups were challenged 6 weeks after administration of theboost vaccine.

FIG. 50 shows the results of the second study. In both groups,substantial increases in anti-Ebola GP were Obtained. At Day 18 afterchallenge with live virus, survival for saline control animals was 0%.In contrast both animals in each of Groups F and G survived,establishing that the vaccine compositions were protective.

DETAILED DESCRIPTION

Disclosed herein are nanoparticles for inducing immune responses,methods for producing and administering them and vaccine compositionscontaining them. The nanoparticle provides antigen surrounding andassociated with a detergent core that result in a structure thatprovides enhanced stability by numerous measures. The detergent core andantigen associate via a physico-chemical interaction mediated by theproperties of the antigen and detergent. In addition, the nanoparticlesoffer especially good antigen presentation to immune systems which,without being bound by theory, is thought to result from the orientationof the antigens around the detergent core.

In one aspect, the disclosure provides compositions containingrecombinant viral glycoprotein nanoparticles. In particular aspects, theglycoproteins are recombinantly expressed in a suitable host cell. Inone embodiment, the host cell is an insect cell. In an exemplaryembodiment, the insect cell is an Sf9 cell.

In particular aspects, the disclosure provides immunogenic compositionscomprising one or more viral glycoprotein species in a nanoparticlestructure where the glycoprotein is in the form of a trimer and eachnanoparticle contains at least one trimer associated with a non-ionicdetergent core. In particular aspects, a nanoparticle consists of anantigen, such as a viral glycoprotein, from only one pathogen.

The nanoparticles may be used for the prevention and/or treatment ofviral infection. Thus, in another aspect, the disclosure provides amethod for eliciting an immune response against a virus. The methodinvolves administering an immunologically effective amount of acomposition containing a nanoparticle to a subject.

The disclosure provides vaccine compositions comprising thenanoparticle. Compositions may contain nanoparticles having antigensfrom multiple pathogens. In some aspects, the vaccine composition maycontain nanoparticles with antigens from more than one viral strain fromthe same species of virus. In aspects, the vaccine composition maycontain nanoparticles with antigens from different virus species. Inanother embodiment, the disclosures provide for a pharmaceutical pack orkit comprising one or more containers filled with one or more of thecomponents of the vaccine compositions.

In another embodiment, the disclosure provides a method of formulating avaccine composition that induces immunity to an infection or at leastone disease symptom thereof to a mammal, comprising adding to thecomposition an effective dose of a nanoparticle. The disclosednanoparticles are useful for preparing compositions that stimulate animmune response that confers immunity or substantial immunity toinfectious agents. Thus, in one embodiment, the disclosure provides amethod of inducing immunity to infections or at least one diseasesymptom thereof in a subject, comprising administering at least oneeffective dose of a nanoparticle.

In some embodiments, the nanoparticles are administered with anadjuvant. In other aspects, the nanoparticles are administered withoutan adjuvant. In some aspects, the adjuvant may be bound to thenanoparticle, such as by a non-covalent interaction. In other aspects,the adjuvant is co-administered with the nanoparticle but the adjuvantand nanoparticle do not interact substantially.

Also provided herein are methods of manufacturing the nanoparticles andvaccine compositions. Advantageously, the methods provide nanoparticlesthat are substantially free from contamination by other proteins, suchas proteins associated with recombinant expression of proteins inbaculovirus/Sf9 systems.

Definitions

As used herein, and in the appended claims, the singular forms “a”,“an”, and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a protein” canrefer to one protein or to mixtures of such protein, and reference to“the method” includes reference to equivalent steps and/or methods knownto those skilled in the art, and so forth.

As used herein, the term “adjuvant” refers to a compound that, when usedin combination with an immunogen, augments or otherwise alters ormodifies the immune response induced against the immunogen. Modificationof the immune response may include intensification or broadening thespecificity of either or both antibody and cellular immune responses.

As used herein, the term “about” or “approximately” when preceding anumerical value indicates the value plus or minus a range of 10%. Forexample, “about 100” encompasses 90 and 110.

As used herein, the terms “immunogen,” “antigen,” and “epitope” refer tosubstances such as proteins, including glycoproteins, and peptides thatare capable of eliciting an immune response.

As used herein, an “immunogenic composition” is a composition thatcomprises an antigen where administration of the composition to asubject results in the development in the subject of a humoral and/or acellular immune response to the antigen.

As used herein, a “subunit” composition, for example a vaccine, thatincludes one or more selected antigens but not all antigens from apathogen. Such a composition is substantially free of intact virus orthe lysate of such cells or particles and is typically prepared from atleast partially purified, often substantially purified immunogenicpolypeptides from the pathogen. The antigens in the subunit compositiondisclosed herein are typically prepared recombinantly, often using abaculovirus system.

As used herein, “substantially” refers to isolation of a substance (e.g.a compound, polynucleotide, or polypeptide) such that the substanceforms the majority percent of the sample in which it is contained. Forexample, in a sample, a substantially purified component comprises 85%,preferably 85%90%, more preferably at least 95%-99.5%, and mostpreferably at least 99% of the sample. If a component is substantiallyreplaced the amount remaining in a sample is less than or equal to about0.5% to about 10%, preferably less than about 0.5% to about 1.0%

The terms “treat,” “treatment,” and “treating,” as used herein, refer toan approach for obtaining beneficial or desired results, for example,clinical results. For the purposes of this disclosure, beneficial ordesired results may include inhibiting or suppressing the initiation orprogression of an infection or a disease; ameliorating, or reducing thedevelopment of, symptoms of an infection or disease; or a combinationthereof.

“Prevention,” as used herein, is used interchangeably with “prophylaxis”and can mean complete prevention of an infection or disease, orprevention of the development of symptoms of that infection or disease;a delay in the onset of an infection or disease or its symptoms; or adecrease in the severity of a subsequently developed infection ordisease or its symptoms.

As used herein an “effective dose” or “effective amount” refers to anamount of an immunogen sufficient to induce an immune response thatreduces at least one symptom of pathogen infection. An effective dose oreffective amount may be determined e.g., by measuring amounts ofneutralizing secretory and/or serum antibodies, e.g., by plaqueneutralization, complement fixation, enzyme-linked immunosorbent(ELISA), or microneutralization assay.

As used herein, the term “vaccine” refers to an immunogenic composition,such as an immunogen derived from a pathogen, which is used to induce animmune response against the pathogen that provides protective immunity(e.g., immunity that protects a subject against infection with thepathogen and/or reduces the severity of the disease or condition causedby infection with the pathogen). The protective immune response mayinclude formation of antibodies and/or a cell-mediated response.Depending on context, the term “vaccine” may also refer to a suspensionor solution of an immunogen that is administered to a vertebrate toproduce protective immunity.

As used herein, the term “subject” includes humans and other animals.Typically, the subject is a human. For example, the subject may be anadult, a teenager, a child (2 years to 14 years of age), an infant (1month to 24 months), or a neonate (up to 1 month). In some aspects, theadults are seniors about 65 years or older, or about 60 years or older.In some aspects, the subject is a pregnant woman or a woman intending tobecome pregnant. In other aspects, subject is not a human; for example anon-human primate; for example, a baboon, a chimpanzee, a gorilla, or amacaque. In certain aspects, the subject may be a pet, such as a dog orcat.

As used herein, the term “pharmaceutically acceptable” means beingapproved by a regulatory agency of a U.S. Federal or a state governmentor listed in the U.S. Pharmacopeia, European Pharmacopeia or othergenerally recognized pharmacopeia for use in mammals, and moreparticularly in humans. These compositions can be useful as a vaccineand/or antigenic compositions for inducing a protective immune responsein a vertebrate.

As used herein, the term “about” means plus or minus 10% of theindicated numerical value.

Overview

Antigens derived from pathogens are combined with non-ionic detergentsto provide nanoparticles surrounding a detergent core that have improvedstability and excellent immunogenicity. The disclosure also provides formethods and compositions for vaccinating a subject against pathogens. Inparticular aspects, the pathogen is a virus. The antigen is typically aprotein, often a glycoprotein. Also disclosed are compositionscontaining the nanoparticles which find use as vaccine compositions.Methods of producing the nanoparticles and producing the vaccinecompositions are also disclosed.

Nanoparticle Structure and Morphology

Nanoparticles of the present disclosure comprise antigens associatedwith non-ionic detergent core. FIG. 6 upper panel illustrates an exampleof multiple RSV F antigens associated with the detergent core. FIG. 35shows Ebola nanoparticles. Advantageously, the nanoparticles haveimproved resistance to environmental stresses such that they provideenhanced stability.

In particular embodiments, the nanoparticles are composed of multipleprotein trimers surrounding a non-ionic detergent core. For example,each nanoparticle may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or15 trimers. Typically, each nanoparticle contains 2 to 9 trimers. Inparticular embodiments, each nanoparticle contains 2 to 6 trimers.Compositions disclosed herein may contain nanoparticles having differentnumbers of timers. For example, a composition may contain nanoparticleswhere the number of trimers ranges from 2-9; in other embodiments, thenanoparticles in a composition may contain from 2-6 trimers. Inparticular embodiments, the compositions contain a heterogeneouspopulation of nanoparticles having 2 to 6 trimers per nanoparticle, or 2to 9 trimers per nanoparticle. In other embodiments, the compositionsmay contain a substantially homogenous population of nanoparticles. Forexample, the population may contain about 95% nanoparticles having 5trimers.

The antigens are associated with the non-ionic detergent-containing coreof the nanoparticle. Typically, the detergent is selected frompolysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60),polysorbate-65 (PS65) and polysorbate-80 (PS80). The presence of thedetergent facilitates formation of the nanoparticles by forming a corethat organizes and presents the antigens. Thus, in certain embodiments,the nanoparticles may contain the antigens assembled intomulti-oligomeric glycoprotein-PS80 protein-detergent nanoparticles withthe head regions projecting outward and hydrophobic regions and PS80detergent forming a central core surrounded by the antigens.

The nanoparticles disclosed herein range in Z-ave size from about 20 nmto about 60 nm, about 20 nm to about 50 nm, about 20 nm to about 45 nm,or about 25 nm to about 45 nm. Particle size (Z-ave) is measured bydynamic light scattering (DLS) using a Malvern Zetasizer, unlessotherwise specified.

Several nanoparticle types may be included in vaccine compositionsdisclosed herein. In some aspects, the nanoparticle type is in the formof an anisotropic rod, which may be a dimer or a monomer. In otheraspects, the nanoparticle type is a spherical oligomer. In yet otheraspects, the nanoparticle may be described as an intermediatenanoparticle, having sedimentation properties intermediate between thefirst two types. Formation of nanoparticle types may be regulated bycontrolling detergent and protein concentration during the productionprocess. Nanoparticle type may be determined by measuring sedimentationco-efficient. See FIG. 9A and 9B, for examples showing RSV Fnanoparticles. See also, FIG. 8 illustrating control over nanoparticlesize by adjusting detergent and protein concentrations.

Nanoparticle Production

The nanoparticles of the present disclosure are non-naturally occurringproducts, the components of which do not occur together in nature.Generally, the methods disclosed herein use a detergent exchangeapproach wherein a first detergent is used to isolate a protein and thenthat first detergent is exchanged for a second detergent to form thenanoparticles.

The antigens contained in the nanoparticles are typically produced byrecombinant expression in host cells. Standard recombinant techniquesmay be used. Typically, the proteins are expressed in insect host cellsusing a baculovirus system. In preferred embodiments, the baculovirus isa cathepsin-L knock-out baculovirus. In other preferred embodiments, thebaculovirus is a chitinase knock-out baculovirus. In yet other preferredembodiments, the baculovirus is a double knock-out for both cathepsin-Land chitinase. High level expression may be obtained in insect cellexpression systems Non limiting examples of insect cells are, Spodopterafrugiperda (Si) cells, e.g. Sf9, Sf21, Trichoplusia ni cells, e.g. HighFive cells, and Drosophila S2 cells.

Typical transfection and cell growth methods can be used to culture thecells. Vectors, e.g., vectors comprising polynucleotides that encodefusion proteins, can be transfected into host cells according to methodswell known in the art. For example, introducing nucleic acids intoeukaryotic cells can be achieved by calcium phosphate co-precipitation,electroporation, microinjection, lipofection, and transfection employingpolyamine transfection reagents. In one embodiment, the vector is arecombinant baculovirus.

Methods to grow host cells include, but are not limited to, batch,batch-fed, continuous and perfusion cell culture techniques. Cellculture means the growth and propagation of cells in a bioreactor (afermentation chamber) where cells propagate and express protein (e.g.recombinant proteins) for purification and isolation. Typically, cellculture is performed under sterile, controlled temperature andatmospheric conditions in a bioreactor. A bioreactor is a chamber usedto culture cells in which environmental conditions such as temperature,atmosphere, agitation and/or pH can be monitored. In one embodiment, thebioreactor is a stainless steel chamber. In another embodiment, thebioreactor is a pre-sterilized plastic bag (e.g. Cenbag®, Wave Biotech,Bridgewater, N.J.). In other embodiment, the pre-sterilized plastic bagsare about 50 L to 3500 L bags.

Detergent Extraction and Purification of Nanoparticles

After growth of the host cells, the protein may be harvested from thehost cells using detergents and purification protocols. Once the hostcells have grown for 48 to 96 hours, the cells are isolated from themedia and a detergent-containing solution is added to solubilize thecell membrane, releasing the protein in a detergent extract. TritonX-100 and tergitol, also known as NP-9, are each preferred detergentsfor extraction. The detergent may be added to a final concentration ofabout 0.1% to about 1.0%. For example, the concentration may be about0.1%, about 0.2%, about 0.3%, about 0.5%, about 0.7%, about 0.8%, orabout 1.0%. In certain embodiments, the range may be about 0.1% to about0.3%. Preferably, the concentration is about 0.5%.

In other aspects, different first detergents may be used to isolate theprotein from the host cell. For example, the first detergent may beBis(polyethylene glycol bis[imidazoylcarbonyl]), nonoxynol-9,Bis(polyethylene glycol bis[imidazoyl carbonyl]), Brij® 35, Brij®56,Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P, Cremophor® EL,Decaethyleneglycol monododecyl ether, N-Decanoyl-N-methylglucamine,n-Decyl alpha-Dglucopyranoside, Decyl beta-D-maltopyranoside,n-Dodecanoyl-N-methylglucamide, nDodecyl alpha-D-maltoside, n-Dodecylbeta-D-maltoside, n-Dodecyl beta-D-maltoside, Heptaethylene glycolmonodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethyleneglycol monotetradecyl ether, n-Hexadecyl beta-D-maltoside, Hexaethyleneglycol monododecyl ether, Hexaethylene glycol monohexadecyl ether,Hexaethylene glycol monooctadecyl ether, Hexaethylene glycolmonotetradecyl ether, Igepal CA-630,Igepal CA-630,Methyl-6-0-(N-heptylcarbamoyl)-alpha-D-glucopyranoside, Nonaethyleneglycol monododecyl ether, N-Nonanoyl-N-methylglucamine,N-NonanoylN-methylglucamine, Octaethylene glycol monodecyl ether,Octaethylene glycohnonododecyl ether, Octaethylene glycol monohexadecylether, Octaethylene glycol monooctadecyl ether, Octaethylene glycolmonotetradecyl ether, Octyl-beta-D glucopyranoside, Pentaethylene glycolmonodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethyleneglycol monohexadecyl ether, Pentaethylene glycol monohexyl ether,Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctylether, Polyethylene glycol diglycidyl ether, Polyethylene glycol etherW-1, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate,Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether,Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate,Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl),Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillajabark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85,Tergitol Type 15-S-12, Tergitol Type 15-S-30, Tergitol Type 15-S-5,Tergitol Type 15-S-7, Tergitol Type 15-S-9, Tergitol Type NP-10,Tergitol Type NP-4, Tergitol Type NP-40, Tergitol, Type NP-7 TergitolType NP-9, Tergitol Type TMN-10, Tergitol Type TMN-6, Triton X-100 orcombinations thereof.

The nanoparticles may then be isolated from cellular debris usingcentrifugation. In some embodiments, gradient centrifugation, such asusing cesium chloride, sucrose and iodixanol, may be used. Othertechniques may be used as alternatives or in addition, such as standardpurification techniques including, e.g., ion exchange, affinity, and gelfiltration chromatography.

For example, the first column may be an ion exchange chromatographyresin, such as Fractogel® EMD TMAE (EMD Millipore), the second columnmay be a lentil (Lens culinaris) lectin affinity resin, and the thirdcolumn may be a cation exchange column such as a Fractogel® EMD SO3(EMT) Millipore) resin. In other aspects, the cation exchange column maybe an MMC column or a Nuvia. C Prime column (Bio-Rad Laboratories, Inc).Preferably, the methods disclosed herein do not use a detergentextraction column; for example a hydrophobic interaction column. Such acolumn is often used to remove detergents during purification but maynegatively impact the methods disclosed here.

Detergent Exchange

To form nanoparticles, the first detergent, used to extract the proteinfrom the host cell is substantially replaced with a second detergent toarrive at the nanoparticle structure. NP-9 is a preferred extractiondetergent. Typically, the nanoparticles do not contain detectable NP-9when measured by HPLC. The second detergent is typically selected fromthe group consisting of PS20, PS40, PS60, PS65, and PS80. Preferably,the second detergent is PS80. To maintain the stability of thenanoparticle formulations, the ratio of the second detergent and proteinis maintained within a certain range.

In particular aspects, detergent exchange is performed using affinitychromatography to bind glycoproteins via their carbohydrate moiety. Forexample, the affinity chromatography may use a legume lectin column.Legume lectins are proteins originally identified in plants and found tointeract specifically and reversibly with carbohydrate residues. See,for example, Sharon and Lis, “Legume lectins—a large family ofhomologous proteins,” FASEB J. 1990 November; 4(14):3198-208; Liener,“The Lectins: Properties, Functions, and Applications in Biology andMedicine,” Elsevier, 2012. Suitable lectins include concanavalin A (conA), pea lectin, sainfoin lect, and lentil lectin. Lentil lectin is apreferred column for detergent exchange due to its binding properties.See, for instance, Example 10. Lectin columns are commerciallyavailable; for example, Capto Lentil Lectin, is available from GEHealthcare. In certain aspects, the lentil lectin column may use arecombinant lectin. At the molecular level, it is thought that thecarbohydrate moieties bind to the lentil lectin, freeing the amino acidsof the protein to coalesce around the detergent resulting in theformation of a detergent core providing nanoparticles having multiplecopies of the antigen, e.g., glycoprotein oligomers which can be dimers,trimers, or tetramers anchored in the detergent.

The detergent, when incubated with the protein to form the nanoparticlesdud ng detergent exchange, may be present at up to about 0.1% (w/v)during early purifications steps and this amount is lowered to achievethe final nanoparticles having optimum stability. For example, thenon-ionic detergent (e.g., PS80) may be about 0.03% to about 0.1%.Preferably, for improved stability, the nanoparticle contains about0.03% to about 0.05% PS80. Amounts below about 0.03% PS80 informulations do not show as good stability. Further, if the PS80 ispresent above about 0.05%, aggregates are formed. Accordingly, about0.03% to about 0.05% PS80 provides structural and stability benefitsthat allow for long-term stability of nanoparticles with reduceddegradation.

Detergent exchange may be performed with proteins purified as discussedabove and purified, frozen for storage, and then thawed for detergentexchange.

Enhanced Stability and Enhanced Immunogenicity of Nanoparticles

Without being bound by theory, it is thought that associating theantigen with a non-ionic detergent core offers superior stability andantigen presentation. The nanoparticles disclosed herein providesurprisingly good stability and immunogenicity. Advantageous stabilityis especially useful for vaccines used in countries lacking properstorage; for example, certain locations in Africa may lack refrigerationand so vaccines for diseases prevalent in areas facing difficult storageconditions, such as Ebola virus and RSV, benefit particularly fromimproved stability. Further, the HA influenza nanoparticles producedusing the neutral pH approach exhibit superior folding to knownrecombinant flu vaccines.

Notably, prior approaches to using detergents to produce RSV vaccinesincluding split vaccines such as described in US 2004/0028698 to Colauet al. failed to produce effective structures. Rather than nanoparticleshaving proteins surrounding a detergent core as disclosed herein, Colauet al's compositions contained amorphous material lacking identifiableviral structures, presumably resulting in failure to present epitopes tothe immune system effectively. In addition, the disclosed nanoparticleshave particularly enhanced stability because the orientation of theantigens, often glycoproteins, around the detergent core stericallyhinders access of enzymes and other chemicals that cause proteindegradation.

The nanoparticles have enhanced stability as determined by their abilityto maintain immunogenicity after exposure to varied stress. Stabilitymay be measured in a variety of ways. In one approach, a peptide map maybe prepared to determine the integrity of the antigen protein aftervarious treatments designed to stress the nanoparticles by mimickingharsh storage conditions. Thus, a measure of stability is the relativeabundance of antigen peptides in a stressed sample compared to a controlsample. FIG. 12 shows that even after various different stresses to anRSV F nanoparticle composition, robust immune responses are achieved.FIG. 13 illustrates the improved protease resistance provided by thenanoparticles using PS80 levels above 0.015%. Notably, at 18 months PS80at 0.03% shows a 50% reduction in formation of truncated speciescompared to 0.015% PS80. The nanoparticles disclosed herein are stableat 2-8° C. Advantageously, however, they are also stable at 25° C. forat least 2 months. In some embodiments, the compositions are stable at25° C. for at least 3 months, at least 6 months, at least 12 months, atleast 18 months, or at least 24 months. For RSV-F nanoparticles,stability may be determined by measuring formation of truncated F1protein, as shown in FIG. 13 . Advantageously, the RSV-F nanoparticlesdisclosed herein advantageously retain an intact antigenic site II at anabundance of 90 to 100% as measured by peptide mapping compared to thecontrol RSV-F protein in response to various stresses including pH(pH3.7), high pH (pH10), elevated temperature (50° C. for 2 weeks), andeven oxidation by peroxide as shown in FIG. 12 .

It is thought that the position of the glycoprotein anchored into thedetergent core provides enhanced stability by reducing undesirableinteractions. For example, the improved protection againstprotease-based degradation may be achieved through a shielding effectwhereby anchoring the glycoproteins into the core at the molar ratiosdisclosed herein results in steric hindrance blocking protease access.

Thus, in particular aspects, disclosed herein are RSV-F nanoparticles,and compositions containing the same, that retain 90% to 100%, of intactSite II peptide, compared to untreated control, in response to one ormore treatments selected from the group consisting of incubation at 50°C. for 2 weeks, incubation at pH 3.7 for 1 week at 25° C., incubation atpH 10 for 1 week at 25° C., agitation for 1 week at 25° C., andincubation with an oxidant, such as hydrogen peroxide, for 1 week at 25°C. Additionally, after such treatments, the compositions functionalityis retained. See FIGS. 12A-12D. For example, neutralizing antibody,anti-RSV IgG and PCA titers are preserved compared to control.

Enhanced immunogenicity is exemplified by the cross-neutralizationachieved by the influenza nanoparticles. It is thought that theorientation of the influenza antigens projecting from the core providesa more effective presentation of epitopes to the immune system.

Nanoparticle Antigens

In typical embodiments, the antigens used to produce the nanoparticlesare viral proteins. In some aspects, the proteins may be modified butretain the ability to stimulate immune responses against the naturalpeptide. In some aspects, the protein inherently contains or is adaptedto contain a transmembrane domain to promote association of the proteininto a detergent core. Often the protein is naturally a glycoprotein.

RSV Antigens

In one aspect, the virus is Respiratory Syncytial Virus (RSV) and theviral antigen is the Fusion glycoprotein. The structure and function ofRSV F proteins is well characterized. See FIG. 1 , for an example ofwild-type structure. Suitable RSV-F proteins for use in the compositionsdescribed herein can be derived from RSV strains such as A2, Long, ATCCVR-26, 19, 6265, E49, E65, B65, RSB89-6256, RSB89-5857, RSB89-6190, andRSB89-6614. In certain embodiments, RSV F proteins are mutated comparedto their natural variants. These mutations confer desirablecharacteristics, such as improved protein expression, enhancedimmunogenicity and the like. Additional information describing RSV-Fprotein structure can be found at Swanson et al. A Monomeric UncleavedRespiratory Syncytial Virus F Antigen Retains Profusion-SpecificNeutralizing Epitopes. Journal of Virology, 2014, 88, 11802-11810. JasonS. McLellan et al. Structure of RSV Fusion Glycoprotein Trimer Bound toa Perfusion-Specific Neutralizing Antibody. Science, 2013, 340,1113-1117.

The primary fusion cleavage is located at residues 131 to 136corresponding to SEQ ID NO:2. Inactivation of the primary fusioncleavage site may be achieved by mutating residues in the site, with theresult that furin can no longer recognize the consensus site. Forexample, inactivation of the primary furin cleavage site may beaccomplished by introducing at least one amino acid substitution atpositions corresponding to arginine 133, arginine 135, and arginine 136of the wild-type RSV F protein (SEQ ID NO:2). In particular aspects,one, two, or all three of the arginines are mutated to glutamine. Inother aspects, inactivation is accomplished by mutating the wild-typesite to one of the following sequences: KKQKQQ (SEQ ID NO: 14), QKQKQQ(SEQ ID NO:15), KKQKRQ (SEQ ID NO: 16), and GRRQQR (SEQ ID NO: 17).

In particular aspects, from 1 to 10 amino acids of the corresponding toacids 137 to 145 of SEQ ID NO: 2 may be deleted, including theparticular examples of suitable RSV F proteins shown below. Each of SEQID NOS 3-13 may optionally be prepared with an active primary fusioncleavage site KKRKRR. (SEQ ID NO:18). The wild type strain in SEQ IDNO:2 has sequencing errors (A to P, V to I, and V to M) that arecorrected in SEQ ID NOS:3-13. Following expression of the RSV-F proteinin a host cell, the N-terminal signal peptide is cleaved to provide thefinal sequences. Typically, the signal peptide is cleaved by host cellproteases. In other aspects, however, the full-length protein may beisolated from the host cell and the signal peptide cleaved subsequently.The N-terminal RSV F signal peptide consists of amino acids of SEQ IDNO: 26 (MELLILKANAITTILTAVTFCFASG). Thus, for example, followingcleavage of the signal peptide from SEQ ID NO:8 during expression andpurification, a mature protein having the sequence of SEQ ID NO: 19 isobtained and used to produce a RSV F nanoparticle vaccine. See FIG. 1B.Optionally, one or more up to all of the RSV F signal peptide aminoacids may be deleted, mutated, or the entire signal peptide may bedeleted and replaced with a different signal peptide to enhanceexpression. An initiating methionine residue is maintained to initiateexpression.

Expressed Protein Primary Fusion SEQ ID NO: Fusion Domain DeletionCleavage Site sequence  1 Wild type Strain A2 (nucleic) KKRKRR (active) 2 Wild type Strain A2 (protein) KKRKRR (active)  3 Deletion of 137 (Δ1)KKQKQQ (inactive)  4 Deletion of 137-138 (Δ2) KKQKQQ (inactive)  5Deletion of 137-139 (Δ3) KKQKQQ (inactive)  6 Deletion of 137-140 (Δ4)KKQKQQ (inactive)  7 Deletion of 137-141 (Δ5) KKQKQQ (inactive)  8Deletion of 137-146 (Δ10) KKQKQQ (inactive)  9 Deletion of 137-142 (Δ6)KKQKQQ (inactive) 10 Deletion of 137-143 (Δ7) KKQKQQ (inactive) 11Deletion of 137-144 (Δ8) KKQKQQ (inactive) 12 Deletion of 137-145 (Δ9)KKQKQQ (inactive) 13 Deletion of 137-145 (Δ9) KKRKRR (active)

In some aspects, the RSV F protein disclosed herein is only altered froma wild-type strain by deletions in the fusion domain, optionally withinactivation of the primary cleavage site. In other aspects, additionalalterations to the RSV F protein may be made. Typically, the cysteineresidues are mutated. Typically, the N-linked glycosylation sites arenot mutated. See FIG. 1B. Additionally, the antigenic site II, alsoreferred to herein as the Palivizumab site because of the ability of thepalivizumab antibody to bind to that site, is preserved. The Motavizumabantibody also binds at site II. Additional suitable RSV-F proteins,incorporated by reference, are found in U.S Publication US 2011/0305727,including in particular, RSV-F proteins containing the sequencesspanning residues 100 to 150 as disclosed in FIG. 1C therein.

In certain other aspects, the RSV F1 or F2 domains may havemodifications relative to the wild-type strain as shown in SEQ ID NO:2.For example, the F1 domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10alterations, which may be mutations or deletions. Similarly, the F2domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations, which maybe mutations or deletions. The F 1 and domains may each independentlyretain at least 90%, at least 94% at least 95% at least 96% at least 98%at least 99%, or 100% identity to the wild-type sequence.

In a particular example, an RSV nanoparticle drug product may containabout 0.025% to about 0.03% PS80 with RSV F at a range of about 270μg/mL to about 300 μg/mL, or about 60 μg/mL to about 300 μg/mL. In otheraspects, the nanoparticle drug product may contain about 0.035% to about0.04% PS80 in a composition with RSV F at 300 μg/mL to about 500 μg/mL.In yet other aspects, the nanoparticle drug product may contain about0.035% to about 0.04% PS80 in a composition with RSV F at 350-500 μg/mL.

Because the concentrations of antigen and detergent can vary, theamounts of each may be referred as a molar ratio of non-ionic detergent:protein. For example, the molar ratio of PS80 to protein is calculatedby using the PS80 concentration and protein concentration of the antigenmeasured by ELISA/A280 and their respective molecular weights. Themolecular weight of PS80 used for the calculation is 1310 and, using RSVF as an example, the molecular weight for RSV F is 65 kD. Molar ratio iscalculated as a follows: (PS80 concentration×10×65000)÷(1310×RSV Fconcentration in mg/mL). Thus, for example, as shown FIG. 13 , thenanoparticle concentration, measured by protein, is 270 μg/mL and thePS80 concentrations are 0.015% and 0.03%. These have a molar ratio ofPS80 to RSV F protein of 27:1 (that is, 0.015×10×65000/(1310×0.27)) and55:1, respectively.

In particular aspects, the molar ratio is in a range of about 30:1 toabout 80:1, about 30:1 to about 70:1, about 30:1 to about 60:1, about40:1 to about 70:1, or about 40:1 to about 50:1. Often, the replacementnon-ionic detergent is PS80 and the molar ratio is about 30:1 to about50:1, PS80: protein. For RSV-F glycoprotein, nanoparticles having amolar ratio in a range of 35:1 to about 65:1, and particularly a ratioof about 45:1, are especially stable.

Influenza Antigens

The nanoparticle platform is especially useful for presenting influenzaantigens to the immune system of a subject. Previous approaches toproducing influenza nanoparticle vaccines have used hydrophobicinteraction columns to remove detergent or have contained only minimalamounts of detergent to reduce non-specific interactions that aroseduring product purification. It has now been discovered, however, thatby performing a detergent exchange step nanoparticles having a non-ionicdetergent core having excellent properties can be produced. Thenanoparticles show excellent stability as evidenced by resistance todegradation by environmental stresses, which permits extended storageperiods, as especially useful property for vaccines. In addition, thenanoparticle structure is such that it presents the antigens in aparticularly advantageous fashion.

The influenza nanoparticles are especially useful as vaccines as theantibodies they induce contain broadly neutralizing antibodies. Thus,antibodies induced by a nanoparticle administered. in one year canneutralize influenza viral strains arising from the “drift” process insubsequent years. It is thought that these epitopes that induce thesebroadly neutralizing antibodies have not been exposed at all, or exposedeffectively, in prior influenza vaccines, or that the epitopes wereinsufficiently stable in prior formulations. The nanoparticles disclosedherein resolve those problems by presenting cross-protective epitopesanchored around a non-ionic detergent core with enhanced stability.

Finally, the methods disclosed herein provide for especially high yieldinfluenza nanoparticles with good purity, which is advantageouseconomically in general and especially valuable for viruses that requirerapid production of large amounts, such as pandemic influenza virus.

In certain embodiments, a nanoparticle may contain an HA or an NAprotein. For example, a nanoparticle may contain a HA protein selectedfrom the sub-types H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12,H13, H14, H15, and H16. A nanoparticle may contain an NA proteinselected from the sub-types N1, N2, N3, N4, N5, N6, N7, N8 and N9.Phylogenetically, the HA and NA proteins are split into groups. For HA,Group 1 contains H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16, andgroup 2 contains H3, H4, H7, H10, H14, and H15. NAs also form twogroups: Group 1 contains N1, N4, N5, and N8, and Group 2 contains N2,N3, N6, N7, and N9. In certain aspects, the antigen may have at least90% identity, at least 95% identity, at least 97% identity, or at least99% to the native influenza HA protein or the NA protein.

The HA and NA proteins used for the nanoparticles are typicallyfull-length sequences. In certain aspects, portions of the C-terminusmay be removed.

Advantageously, compositions having influenza can induce responsesagainst heterologous strains of influenza, even when additional pathogennanoparticles disclosed herein are co-administered. By inducingresponses against heterologous influenza strains, broader protection isachieved. Thus, in particular aspects, the homologous HAI titer inducedwith Matrix M-adjuvanted compositions is about 800 to about 2000. Inparticular aspects, the heterologous HAI titer is about 1300. Inparticular aspects, the heterologous HAI titer induced with MatrixM-adjuvanted compositions is about 200 to about 400; for example, theheterologous HAI titer may be about 300.

In certain aspects, the influenza nanoparticles are trypsin-resistantnanoparticles produced using neutral pH purification. Trypsin resistanceis achieved by neutral pH range of above 6.8 to 8.5 during purificationand formulation of the HA nanoparticles. In particular aspects, the pHrange during purification and formulation of the HA nanoparticles is 7.0to 8.5, 7.0 to 7.5, or 7.2 to 7.5. HA nanoparticle stability may bemeasured by Differential Scanning calorimetry (DSC). DSC measures thethermodynamic profile of macromolecules in solution, specifically, bymeasuring the difference in heat energy uptake between a sample solutionand appropriate reference (buffer/solvent) by varying the temperature ina controlled manner. DSC provides data as transition midpoints (Tm),defined as temperatures where half the protein is denatured/unfolded andhalf in the native/folded state. In certain aspects, thetrypsin-resistant HA nanoparticles herein have a Tm peak in a range ofabout 60° C. to 75° C.; for example, the Tm may be 60° C., 65° C., 70°C. or 75° C.

Trypsin resistance indicates that the HA protein is properly folded andthus provides a vaccine product having better stability andimmunogenicity. The sensitivity of HA proteins varies fromstrain-to-strain and the neutral pH production disclosed herein thusprovides a process for maximizing immunogenicity for all strains,especially pH sensitive strains. Without being bound by theory it isthought that the combination of the detergent exchange and neutral pHlevels preserve the HA protein in a structure that renders it resistantto proteases, particularly trypsin. Thus, by having the HA proteinassociated around a non-ionic detergent core combined with neutral pHpurification, HA proteins of particularly good stability andimmunogenicity are achieved. In addition, the methods of producing thenanoparticles provide excellent levels of protein for use in a vaccine.In particular aspects, the HA nanoparticles are produced, as measured byA280, at about 10 mg/L of cell culture to about 30 mg/L, or higher, atabout 20 mg/L to about 30 mg/L.

The trypsin-resistant HA nanoparticles may be prepared as described inFIG. 24 . Briefly, the various steps, including detergent exchanges areperformed with buffers above pH 7.0; often in the range of about pH 7.2to about pH 7.4. FIG. 25D provides an example of the betterthermostability achieved with trypsin resistant nanoparticles. The TFFand MMC production lots were obtained using neutral pH whereas themisfolded low pH lot is substantially degraded and/or misfolded.

Ebola Antigens

The disclosure also provided methods and compositions for treating,ameliorating, or preventing Ebola virus infection and/or disease. Inparticular, the compositions are vaccine compositions. Advantageously,the vaccine compositions disclosed herein provide for 100% survival tolethal challenge in animal models. The compositions also maintain aviral load about or below the detectable limit when using RT-PCR todetect viral nucleic acid.

In one aspect, the disclosure provides compositions containingrecombinant Ebola virus Glycoprotein (GP) nanoparticles in combinationwith saponin-based adjuvants.

In particular aspects, the disclosure provides immunogenic compositionscomprising one or Ebola virus GP proteins in a nanoparticle structurewhere the GP protein is in the form of a timer and each nanoparticlecontains at least one trimer attached to a non-ionic detergent core.

The Ebola GP nanoparticles may be used for the prevention and/ortreatment of Ebola infection. In another aspect, the present disclosureprovides pharmaceutically acceptable vaccine compositions comprising anEbola GP nanoparticle. In some aspects, nanoparticles from more than onestrain are in the vaccine. In another embodiment, the disclosuresprovides for a pharmaceutical pack or kit comprising one or morecontainers filled with one or more of the ingredients of the vaccineformulations.

Ebola Glycoproteins

The Ebola antigen use to prepare the nanoparticle is typically an EbolaGlycoprotein (GP) antigen. The antigen may be derived from a variety ofstrains. The compositions disclosed herein may contain nanoparticlesfrom one, two, three, four, five, or six separate Ebola strains. Forexample, the strain may be Makona, Sudan, Zaire, Reston. In otheraspects, the Ebola GP may share amino acid or nucleic acid identity withone or more of these strains. For example, the GP may be about 80%identical, about 85% identical, about 90% identical, about 95%identical, about 97% identical, about 98% identical, or about 99%identical to one or more of the GPs from the Makona, Sudan, Zaire, orReston viruses, wherein identity is measured over the full length of theprotein or nucleic acid. In some aspects, the Ebola GP may comprise, orconsist of, SEQ ID NO:27 or 28, or a protein having identity thereto.

A representative Zaire strain sequence is provided at GenBank AccessionNo. AAB81004 (SEQ ID NO:27). The first underlined portion shows theN-terminus for the GP1 protein. The preceding signal peptide is cleavedoff during processing following expression in the cell prior topurification and formulation into a vaccine. Shown in bold is the furincleavage site. Following the bold text, the N-terminus for the GP2protein is shown. FIG. 7A shows a cartoon of the protein structure.

MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRR EAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVF

The Makona isolate sequence is provided at GenBank Accession No.4JG-4419 (SEQ ID NO:28). As above, the first underlined portion showsthe N-terminus for the GP1 protein. The preceding signal peptide iscleaved off during processing. Shown in bold is the furin cleavage site.Following the bold text, the N-terminus for the GP2 protein is shown.See also FIG. 7B.

MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSVTKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYASGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTAVSNGPKNISGQSPARTSSDPETNTTNEDHKIMASENSSAMVQVHSQGRKAAVSHLTTLATISTSPQPPTTKTGPDNSTHNTPVYKLDISEATQVGQHHRRADNDSTASDTPPATTAAGPLKAENTNTSKSADSLDLATTTSPQNYSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTR REVIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYTEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVF

The ability of the vaccine compositions to stimulate immune responseswas confirmed in three animal models. First, a mouse model was used. Arecombinant EBOV/Mak full length GP nanoparticle vaccine formulated withMatrix-M, AlPO₄ or saline was evaluated. Immunization of mice withnon-adjuvanted or AlPO₄ adjuvanted EBOV/Mak GP induced modest antibodyand cellular responses; however, when adjuvanted with Matrix-M, purifiedEBOV/Mak GP nanoparticles were highly immunogenic and protective in amurine challenge model. Immunization of mice with Matrix-M adjuvantedEBOV/Mak GP resulted in a significant increase in anti-EBOV/Mak GP IgGand Ebola virus neutralizing antibody. Immunization with the Matrix-Madjuvanted EBOV/Mak GP conferred 100% protection from a lethal Ebolavirus challenge while unadjuvanted EBOV/Mak GP was only 10% protectiveand no protection was observed in mice immunized with EBOV/Mak GP withAlPO₄. Thus, in particular aspects, the compositions disclosed hereinprevent Ebola infection.

Co-administration of the EBOV/Mak GP with Matrix-M induced theproduction of a balanced IgG1 and IgG2a subclass response. In theabsence of adjuvant or with AlPO₄, minimal IgG2a antibody was detected.Blaney et al. Antibody quality and protection from lethal Ebola viruschallenge in nonhuman primates immunized with rabies virus basedbivalent vaccine. PLoS Pathog. 2013; 9(5):, showed in a rabies/EBOVchimera vaccine model in non-human primates (NHP) that the antibodyisotype played a role in virus neutralization and protection againstEbola virus challenge. Murine IgG2a antibody is the equivalent of humanIgG1 antibody that binds efficiently to IgG-Fc receptors (FcγR) andcomplement (C1q) (Bruhns, P. Properties of mouse and human IgG receptorsand their contribution to disease models Blood. 2012; 119: 5640-5649;Vidarsson G, Dekkers G, Rispens T. IgG subclasses and allotypes: fromstructure to effector functions. Front. Immunol. 2014; 5:520) and mayhelp resolving viral infections e.g., through antibody-dependentcell-mediated cytotoxicity. All antibodies that were completelyprotective in vivo were of the IgG2a subclass; i.e. the same as humanIgG1. Thus, the compositions disclosed herein stimulate production ofIgG1 antibodies as part of a protective immune response.

The use of the Matrix-M adjuvant provided a dose dependent increase inthe frequency of CD4+ and CD8+ cytokine secreting T cells as well as thenumber of multifunctional T cells producing more than one cytokine. Theobservation that protection from a lethal Ebola virus challenge wasobserved only in the Matrix-M adjuvanted EBOV/Mak GP group correlatedwith the enhanced production of multifunctional T cells.

The use of Matrix-M increased the frequency of GC B cells in the spleenand long lived plasma cells in the bone marrow. GCs are themicro-anatomic locations for B cell differentiation, somatichypermutation, antibody class-switching and formation of memory B cells.Co-administration of the EBOV/Mak GP with the saponin adjuvant Matrix-Malso resulted in an increase of the numbers of T_(FH) cells whichfacilitate GC B cell differentiation and development. The increasedfrequencies of GC and T_(FH) cells induced by Matrix-M adjuvantationcorrelated with the enhanced magnitude of the antibody response and theinduction of a greater numbers of long-lived plasma cell, suggesting theMatrix-M adjuvanted EBOV/Mak GP vaccine may induce an especially durableantibody response.

Each dose of Ebola GP may be combined with adjuvant. AdministeringMatrix-M adjuvant with purified EBOV/Mak GP nanoparticles providesrobust stimulation of the anti-EBOV/Mak GP immune response resulting in100% protective efficacy in the mouse model. The compositions andmethods disclosed herein provide a more rapid onset of anti-EBOV/Mak GPIgG and Ebola virus neutralization antibodies, increased concentrationof IgG2a, as well as increased frequency of multifunctional CD4+ andCD8+ T cells, T_(FH) cells, germinal center B cells and persistence ofEBOV/Mak GP-specific plasma B cells in the bone marrow.

Analysis of the mouse study thus confirms that the compositionsdisclosed herein provided complete protection. To further establish theprotective effect, studies were performed in two separate non-humanprimate models: Baboon and macaques: See Perry et al., “The Baboon(Papio spp.) as a model of human Ebola virus infection,” Viruses. 2012Oct. 23; 4(10):2400-16; Geisbert et al., “Pathogenesis of Ebolahemorrhagic fever in Cynomolgus macaques: evidence that dendritic cellsare early and sustained targets of infection,” Am J Pathol. 2003December; 163(6):2347-70. Accordingly, in some aspects of the disclosurea protective effect includes a reduction on viral load beneath theability of RT-PCR to detect after about 7 days, about 10 days, about 14days, or about 21 days, after virus exposure.

The non-human primate studies further confirmed that the compositionsdisclosed herein are protective. Ebola GP nanoparticles were evaluatedwithout adjuvant and with either Alum or Matrix M adjuvants. See Example23. The immune responses in baboons were extremely robust and sustained.Notably, the inclusion of Matrix M led to a greater immune response thanwith Alum. The results with the macaque model were particularlyunexpected. See Examples 24 and 24. The compositions not only protectedagainst challenge with live Ebola vaccine, the amount of Ebola RNA wasundetectable at Day 10 following challenge with live virus. See FIG. 48. Notably, in one macaque subject, there was a small signal about Day 7;however, by Day 10, levels had returned below the limit of detection. Incontrast, exposure of untreated animals to live Ebola virus resulted ininfection and disease such that the subject was euthanized at Day 9.

Modified Antigens

The antigens disclosed herein encompass variations and mutants of thoseantigens. In certain aspects, the antigen may share identity to adisclosed antigen. Generally, and unless specifically defined in contextof a specifically identified antigens, the percentage identity may be atleast 80%, at least 90%, at least 95%, at least 97%, or at least 98%.Percentage identity can be calculated using the alignment programClustalW2, available at www.ebi.ac.uk/Tools/msa/clustalw2/. Thefollowing default parameters may be used for Pairwise alignment: ProteinWeight Matrix=Gonnet; Gap Open=10; Gap Extension=0.1.

In particular aspects, the protein contained in the nanoparticlesconsists of that protein. In other aspects, the protein contained in thenanoparticles comprise that protein. Additions to the protein itself maybe for various purposes. In some aspects, the antigen may be extended atthe N-terminus, the C-terminus, or both. In some aspects, the extensionis a tag useful for a function, such as purification or detection. Insome aspects the tag contains an epitope. For example, the tag may be apolyglutamate tag, a FLAG-tag, a HA-tag, a polyHis-tag (having about5-10 histidines), a Myc-tag, a Glutathione-S-transferase-tag, a Greenfluorescent protein-tag, Maltose binding protein-tag, a Thioredoxin-tag,or an Fc-tag. In other aspects, the extension may be an N-terminalsignal peptide fused to the protein to enhance expression. While suchsignal peptides are often cleaved during expression in the cell, somenanoparticles may contain the antigen with an intact signal peptide.Thus, when a nanoparticle comprises an antigen, the antigen may containan extension and thus may be a fusion protein when incorporated intonanoparticles. For the purposes of calculating identity to the sequence,extensions are not included.

In some aspects, the antigen may be truncated. For example, theN-terminus may be truncated by about 10 amino acids, about 30 aminoacids, about 50 amino acids, about 75 amino acids, about 100 aminoacids, or about 200 amino acids. The C-terminus may be truncated insteadof or in addition to the N-terminus. For example, the C-terminus may betruncated by about 10 amino acids, about 30 amino acids, about 50 aminoacids, about 75 amino acids, about 100 amino acids, or about 200 aminoacids. For purposes of calculating identity to the protein havingtruncations, identity is measured over the remaining portion of theprotein.

Combination Nanoparticles

A combination nanoparticle, as used herein, refers to a nanoparticlethat induces immune responses against two or more different pathogens.Depending on the particular combination, the pathogens may be differentstrains or sub-types of the same species or the pathogens may bedifferent species. To prepare a combination nanoparticle, glycoproteinsfrom multiple pathogens may be combined into a single nanoparticle bybinding them at the detergent exchange stage. The binding of theglycoproteins to the column followed by detergent exchange permitsmultiple glycoproteins types to form around a detergent core, to providea combination nanoparticle.

The disclosure also provides for vaccine compositions that induce immuneresponses against two or more different pathogens by combining two ormore nanoparticles that each induce a response against a differentpathogen. Optionally, vaccine compositions may contain one or morecombination nanoparticles alone or in combination with additionalnanoparticles with the purpose being to maximize the immune responseagainst multiple pathogens while reducing the number of vaccinecompositions administered to the subject.

Such compositions are particularly desirable when the pathogens areconnected in some aspect. In one example, a composition may containnanoparticles against the strains identified annually by authorities asforming a particular year's seasonal influenza. Typically, for aseasonal influenza vaccine, a vaccine composition contains HA and/or NAnanoparticles that induce immune responses against a strain of three,four, or five influenza sub-types. Thus, different strains of influenzamay be combined in a vaccine composition. In some aspects, thecombination nanoparticle may contain an HA protein from a first strainand an NA protein from a second strain. In other aspects, a nanoparticlemay contain one or more HA and one or more NA proteins from the same ordifferent sub-types. For example, a nanoparticle may contain one or moreHA nanoparticles selected from the sub-types H1, H2, H3, H4, H5, H6, H7,H8, H9, H10, H11, H12, H13, H14, H15 and H16 and/or one or more NAnanoparticles selected from the sub-types N1, N2, N3, N4, N5, N6, N7, N8and N9. Phylogenetically, the HA and NA proteins are split into groups.For HA, Group 1 contains H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16,and group 2 contains H3, H4, H7, H10, H14, and H15. NA proteins alsoform two groups: Group 1 contains N1, N4, N5, and N8, and Group 2contains N2, N3, N6, N7, and N9. In certain aspects, the antigen mayhave at least 90% identity, at least 95% identity, at least 97%identity, or at least 99% to the native influenza HA protein and/or tothe NA protein.

In another example, influenza and RSV both cause respiratory disease andHA, NA, and/or RSV F may therefore be mixed into a combinationnanoparticle or multiple nanoparticles may be combined in a vaccinecomposition to induce responses against RSV and one or more influenzastrains.

Vaccine Compositions

Compositions disclosed herein may be used either prophylactically ortherapeutically, but will typically be prophylactic. Accordingly, thedisclosure includes methods for treating or preventing infection. Themethods involve administering to the subject a therapeutic orprophylactic amount of the immunogenic compositions of the disclosure.Preferably, the pharmaceutical composition is a vaccine composition thatprovides a protective effect. In other aspects, the protective effectmay include amelioration of a symptom associated with infection in apercentage of the exposed population. For example, depending on thepathogen, the composition may prevent or reduce one or more virusdisease symptoms selected from: fever fatigue, muscle pain, headache,sore throat, vomiting, diarrhea, rash, symptoms of impaired kidney andliver function, internal bleeding and external bleeding, compared to anuntreated subject.

The nanoparticles may be formulated for administration as vaccines inthe presence of various excipients, buffers, and the like. For example,the vaccine compositions may contain sodium phosphate, sodium chloride,and/or histidine. Sodium phosphate may be present at about 10 mM toabout 50 mM, about 15 mM to about 25 mM, or about 25 mM; in particularcases, about 22 mM sodium phosphate is present. Histidine may be presentabout 0.1% (w/v), about 0.5% (w/v), about 0.7% (w/v), about 1% (w/v),about 1.5% (w/v), about 2% (w/v), or about 2.5% (w/v). Sodium chloride,when present, may be about 150 mM. In certain compositions, for exampleinfluenza vaccines, the sodium chloride may be present at higheramounts, including about 200 mM, about 30 mM, or about 350 mM.

Certain nanoparticles, particularly RSV F nanoparticles, have improvedstability at slightly acidic pH levels. For example, the pH range forcomposition containing the nanoparticles may be about pH 5.8 to about pH7.0, about pH 5.9 to about pH 6.8, about pH 6.0 to about pH 6.5, aboutpH 6.1 to about pH 6.4, about pH 6.1 to about pH 6.3, or about pH 6.2.Typically, the composition for RSV F protein nanoparticles is about pH6.2. In other nanoparticles, the composition may tend towards neutral;for example, influenza nanoparticles may be about pH 7.0 to pH 7.4;often about pH 7.2.

Adjuvants

In certain embodiments, the compositions disclosed herein may becombined with one or more adjuvants to enhance an immune response. Inother embodiments, the compositions are prepared without adjuvants, andare thus available to be administered as adjuvant-free compositions.Advantageously, adjuvant-free compositions disclosed herein may provideprotective immune responses when administered as a single dose.Alum-free compositions that induce robust immune responses areespecially useful in adults about 60 and older.

Aluminum-Based Adjuvants

In some embodiments, the adjuvant may be alum (e.g. AlPO₄ or Al(OH)₃).Typically, the nanoparticle is substantially bound to the alum. Forexample, the nanoparticle may be at least 80% bound, at least 85% bound,at least 90% bound or at least 95% bound to the alum. Often, thenanoparticle is 92% to 97% bound to the alum in a composition. Theamount of alum is present per dose is typically in a range between about400 μg to about 1250 μg. For example, the alum may be present in a perdose amount of about 300 μg to about 900 μg, about 400 μg to about 800μg, about 500 μg to about 700 μg, about 400 μg to about 600 μg, or about400 μg to about 500 μg. Typically, the alum is present at about 400 μgfor a dose of 120 μg of the protein nanoparticle.

Saponin Adjuvants

Adjuvants containing saponin may also be combined with the immunogensdisclosed herein. Saponins are glycosides derived from the bark of theQuillaja saponaria Molina tree. Typically, saponin is prepared using amulti-step purification process resulting in multiple fractions. Asused, herein, the term “a saponin fraction from Quillaja saponariaMolina” is used generically to describe a semi-purified or definedsaponin fraction of Quillaja saponaria or a substantially pure fractionthereof.

Saponin Fractions

Several approaches for producing saponin fractions are suitable.Fractions A, B, and C are described in U.S. Pat. No. 6,352,697 and maybe prepared as follows. A lipophilic fraction from Quil A, a crudeaqueous Quillaja saponaria Molina extract, is separated bychromatography and eluted with 70% acetonitrile in water to recover thelipophilic fraction. This lipophilic fraction is then separated bysemi-preparative HPLC with elution using a gradient of from 25% to 60%acetonitrile in acidic water. The fraction referred to herein as“Fraction A” or “QH-A” is, or corresponds to, the fraction, which iseluted at approximately 39% acetonitrile. The fraction referred toherein as “Fraction B” or “QH-B” is, or corresponds to, the fraction,which is eluted at approximately 47% acetonitrile. The fraction referredto herein as “Fraction C” or “QH-C” is, or corresponds to, the fraction,which is eluted at approximately 49% acetonitrile. Additionalinformation regarding purification of Fractions is found in U.S. Pat.No. 5,057,540. When prepared as described herein, Fractions A, B and Cof Quillaja saponaria Molina each represent groups or families ofchemically closely related molecules with definable properties. Thechromatographic conditions under which they are obtained are such thatthe batch-to-batch reproducibility in terms of elution profile andbiological activity is highly consistent.

Other saponin fractions have been described. Fractions B3, B4 and B4bare described in EP 0436620. Fractions QA1-QA22 are described EP03632279B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria Molina Spikoside(lsconova AB, Ultunaallén 2B, 756 51 Uppsala, Sweden). Fractions QA-1,QA-2, QA-3, QA 4, QA-5, QA-6, QA-7, QA-8, QA 9, QA-10, QA-11, QA-12,QA-13, QA-14, QA-15, QA-16, QA-17, QA-18, QA-19, QA-20, QA-21, and QA-22of EP 0 3632 279 B2, especially QA-7, QA-17, QA-18, and QA-21 may beused. They are obtained as described in EP 0 3632 279 B2, especially atpage 6 and in Example 1 on page 8 and 9.

The saponin fractions described herein and used for forming adjuvantsare often substantially pure fractions; that is, the fractions aresubstantially free of the presence of contamination from othermaterials. In particular aspects, a substantially pure saponin fractionmay contain up to 40% by weight, up to 30% by weight, up to 25% byweight, up to 20% by weight, up to 15% by weight, up to 10% by weight,up to 7% by weight, up to 5% by weight, up to 2% by weight, up to 1% byweight, up to 0.5% by weight, or up to 0.1% by weight of other compoundssuch as other saponins or other adjuvant materials.

ISCOM Structures

Saponin fractions may be administered in the form of a cage-likeparticle referred to as an ISCOM (Immune Stimulating COMplex). ISCOMsmay be prepared as described in EP0109942B1, EP0242380B1 and EP0180546B 1. In particular embodiments a transport and/or a passenger antigenmay be used, as described in EP 9600647-3 (PCT/SE97/00289).

Matrix Adjuvants

In some aspects, the ISCOM is an ISCOM matrix complex. An ISCOM matrixcomplex comprises at least one saponin fraction and a lipid. The lipidis at least a sterol, such as cholesterol. In particular aspects, theISCOM matrix complex also contains a phospholipid. The ISCOM matrixcomplexes may also contain one or more other immunomodulatory(adjuvant-active) substances, not necessarily a glycoside, and may beproduced as described in EP0436620B1.

In other aspects, the ISCOM is an ISCOM complex. An ISCOM complexcontains at least one saponin, at least one lipid, and at least one kindof antigen or epitope. The ISCOM complex contains antigen associated bydetergent treatment such that that a portion of the antigen integratesinto the particle. In contrast, ISCOM matrix is formulated as anadmixture with antigen and the association between ISCOM matrixparticles and antigen is mediated by electrostatic and/or hydrophobicinteractions.

According to one embodiment, the saponin fraction integrated into anISCOM matrix complex or an ISCOM complex, or at least one additionaladjuvant, which also is integrated into the ISCOM or ISCONI matrixcomplex or mixed therewith, is selected from fraction A, fraction B, orfraction C of Quillaja saponaria, a semi purified preparation ofQuillaja saponaria, a purified preparation of Quillaja saponaria, or anypurified sub-fraction e.g., QA 1-21.

In particular aspects, each ISCOM particle may contain at least twosaponin fractions. Any combinations of weight % of different saponinfractions may be used. Any combination of weight % of any two fractionsmay be used. For example, the particle may contain any weight % offraction A and any weight % of another saponin fraction, such as a crudesaponin fraction or fraction C, respectively. Accordingly, in particularaspects, each ISCOM matrix particle or each ISCOM complex particle maycontain from 0.1 to 99.9 by weight, 5 to 95% by weight, 10 to 90% byweight 15 to 85% by weight, 20 to 80% by weight, 25 to 75% by weight, 30to 70% by weight, 35 to 65% by weight, 40 to 60% by weight, 45 to 55% byweight, 40 to 60% by weight, or 50% by weight of one saponin fraction,e.g. fraction A and the rest up to 100% in each case of another saponine.g. any crude fraction or any other faction e.g. fraction C. The weightis calculated as the total weight of the saponin fractions. Examples ofISCOM matrix complex and ISCOM complex adjuvants are disclosed in U.SPublished Application No. 2013/0129770.

In particular embodiments, the ISCOM matrix or ISCOM complex comprisesfrom 5-99% by weight of one fraction, e.g. fraction A and the rest up to100% of weight of another fraction e.g. a crude saponin fraction orfraction C. The weight is calculated as the total weight of the saponinfractions.

In another embodiment, the ISCOM matrix or ISCOM complex comprises from40% to 99% by weight of one fraction, e.g. fraction A and from 1% to 60%by weight of another fraction, e.g. a crude saponin fraction or fractionC. The weight is calculated as the total weight of the saponinfractions.

In yet another embodiment, the ISCOM matrix or ISCOM complex comprisesfrom 70% to 95% by weight of one fraction e.g., fraction A, and from 30%to 5% by weight of another fraction, e.g., a crude saponin fraction, orfraction C. The weight is calculated as the total weight of the saponinfractions. In other embodiments, the saponin fraction from Quillajasaponaria Molina is selected from any one of QA 1-21.

In addition to particles containing mixtures of saponin fractions, ISCOMmatrix particles and ISCOM complex particles may each be formed usingonly one saponin fraction. Compositions disclosed herein may containmultiple particles wherein each particle contains only one saponinfraction. That is, certain compositions may contain one or moredifferent types of ISCOM-matrix complexes particles and/or one or moredifferent types of ISCOM complexes particles, where each individualparticle contains one saponin fraction from Quillaja saponaria Molina,wherein the saponin fraction in one complex is different from thesaponin fraction in the other complex particles.

In particular aspects, one type of saponin fraction or a crude saponinfraction may be integrated into one ISCOM matrix complex or particle andanother type of substantially pure saponin fraction, or a crude saponinfraction, may be integrated into another ISCOM matrix complex orparticle. A composition or vaccine may comprise at least two types ofcomplexes or particles each type having one type of saponins integratedinto physically different particles.

In the compositions, mixtures of ISCOM matrix complex particles and/orISCOM complex particles may be used in which one saponin fractionQuillaja saponaria Molina and another saponin fraction Quillajasaponaria. Molina are separately incorporated into different ISCOMmatrix complex particles and/or ISCOM complex particles.

The ISCOM matrix or ISCOM complex particles, which each have one saponinfraction, may be present in composition at any combination of weight %.In particular aspects, a composition may contain 0.1% to 99.9% byweight, 5% to 95% by weight, 10% to 90% by weight, 15% to 85% by weight,20% to 80% by weight, 25% to 75% by weight, 30% to 70% by weight, 35% to65% by weight, 40% to 60% by weight, 45% to 55% by weight, 40 to 60% byweight, or 50% by weight, of an ISCOM matrix or complex containing afirst saponin fraction with the remaining portion made up by an ISCOMmatrix or complex containing a different saponin fraction. In someaspects, the remaining portion is one or more ISCOM matrix or complexeswhere each matrix or complex particle contains only one saponinfraction. In other aspects, the ISCOM matrix or complex particles maycontain more than one saponin fraction.

In particular compositions, the saponin fraction in a first ISCOM matrixor ISCOM complex particle is Fraction A and the saponin fraction in asecond ISCOM matrix or ISCOM complex particle is Fraction C.

Preferred compositions comprise a first ISCOM matrix containing FractionA and a second ISCOM matrix containing Fraction C, wherein the FractionA ISCOM matrix constitutes about 70% per weight of the total saponinadjuvant, and the Fraction C ISCOM matrix constitutes about 30% perweight of the total saponin adjuvant. In another preferred composition,the Fraction A ISCOM matrix constitutes about 85% per weight of thetotal saponin adjuvant, and the Fraction C ISCOM matrix constitutesabout 15% per weight of the total saponin adjuvant. Thus, in certaincompositions, the Fraction A ISCOM matrix is present in a range of about70% to about 85%, and Fraction C ISCOM matrix is present in a range ofabout 15% to about 30%, of the total weight amount of saponin adjuvantin the composition. Exemplary QS-7 and QS-21 fractions, their productionand their use is described in U.S. Pat. Nos. 5,057,540; 6,231,859;6,352,697; 6,524,584; 6,846,489; 7,776,343, and 8,173,141, which areincorporated by reference for those disclosures.

Other Adjuvants

In some, compositions other adjuvants may be used in addition or as analternative. The inclusion of any adjuvant described in Vogel et al., “ACompendium of Vaccine Adjuvants and Excipients (2nd Edition),” hereinincorporated by reference in its entirety for all purposes, isenvisioned within the scope of this disclosure. Other adjuvants includecomplete Freund's adjuvant (a non-specific stimulator of the immuneresponse containing killed Mycobacterium tuberculosis), incompleteFreund's adjuvants and aluminum hydroxide adjuvant. Other adjuvantscomprise GMCSP, BCG, MDP compounds, such as thur-MDP and nor-MDP, CGP(MTP-PE), lipid A, and monophosphoryl lipid A (MPL), MF-59, RIBI, whichcontains three components extracted from bacteria, MPL, trehalosedimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween® 80emulsion. In some embodiments, the adjuvant may be a paucilamellar lipidvesicle; for example, Novasomes®. Novasomes® are paucilamellarnonphospholipid vesicles ranging from about 100 nm to about 500 nm. Theycomprise Brij 72, cholesterol, oleic acid and squalene. Novasomes havebeen shown to be an effective adjuvant (see, U.S. Pat. Nos. 5,629,021,6,387,373, and 4,911,928.

Administration and Dosage

Compositions disclosed herein may be administered via a systemic routeor a mucosal route or a transdermal route or directly into a specifictissue. As used herein, the term “systemic administration” includesparenteral routes of administration. In particular, parenteraladministration includes subcutaneous, intraperitoneal, intravenous,intraarterial, intramuscular, or intrasternal injection, intravenous, orkidney dialytic infusion techniques. Typically, the systemic, parenteraladministration is intramuscular injection. As used herein, the term“mucosal administration” includes oral, intranasal, intravaginal,intra-rectal, intra-tracheal, intestinal and ophthalmic administration.Preferably, administration is intramuscular.

Compositions may be administered on a single dose schedule or a multipledose schedule. Multiple doses may be used in a primary immunizationschedule or in a booster immunization schedule. In a multiple doseschedule the various doses may be given by the same or different routese.g., a parenteral prime and mucosal boost, a mucosal prime andparenteral boost, etc. In some aspects, a follow-on boost dose isadministered about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,or about 6 weeks after the prior dose. Typically, however, thecompositions disclosed herein are administered only once yet stillprovide a protective immune response.

In some embodiments, the dose, as measured in pg, may be the totalweight of the dose including the solute, or the weight of the RSV Fnanoparticles, or the weight of the RSV F protein. Dose is measuredusing protein concentration assay either A280 or ELISA.

The dose of antigen, including for pediatric administration, may be inthe range of about 30 μg to about 300 μg, about 90 μg to about 270 μg,about 100 μg to about 160 μg, about 110 μg to about 150 μg, about 120 μgto about 140 μg, or about 140 μg to about 160 μg. In particularembodiments, the dose is about 120 μg, administered with alum. In someaspects, a pediatric dose may be in the range of about 30 μg to about 90μg. Certain populations may be administered with or without adjuvants.For example, when administered to seniors, preferably there is no alum.In certain aspects, compositions may be free of added adjuvant. In suchcircumstances, the dose may be increased by about 10%.

In some embodiments, the dose may be administered in a volume of about0.1 mL to about 1.5 mL, about 0.3 mL to about 1.0 mL, about 0.4 mL toabout 0.6 mL, or about 0.5 mL, which is a typical amount.

In particular embodiments for an RSV vaccine, the dose may comprise anRSV F protein concentration of about 175 μg/mL to about 325 μg/mL, about200 μg/mL to about 300 μg/mL, about 220 μg/mL to about 280 μg/mL, orabout 240 μg/mL to about 260 μg/mL.

All patents, patent applications, references, and journal articles citedin this disclosure are expressly incorporated herein by reference intheir entireties for all purposes.

EXAMPLES Example 1 Expression and Purification of an RSV F Protein

An RSV F protein having SEQ ID NO: 8 was expressed in a baculovirusexpression system and recombinant plaques expressing the RSV F proteinwere picked and confirmed. The recombinant virus was then amplified byinfection of SP), insect cells. A culture of insect cells was infectedat ˜3 MOI (Multiplicity of infection=virus ffu or pfu/cell) withbaculovirus. The culture and supernatant were harvested 48-72 hrspost-infection. The crude cell harvest, approximately 30 mL, wasclarified by centrifugation for 15 minutes at approximately 800×g. Theresulting crude cell harvests containing the RSV F protein were purifiedas described below.

Non-ionic surfactant Tergitol® NP-9 (Nonylphenol Ethoxylate) was used inthe membrane protein extraction protocol. NP-9 was Crude extraction wasfurther purified by passing through anion exchange chromatography,lentil lectin affinity/HIC and cation exchange chromatography. Thewashed cells were lysed by detergent treatment and then subjected to lowpH treatment which leads to precipitation of BV and Sf9 host cell DNAand protein. The neutralized low pH treatment lysate is clarified andfurther purified on anion exchange and affinity chromatography before asecond low pH treatment is performed.

Affinity chromatography was used to remove Sf9/BV proteins, DNA andNP-9, as well as concentrate the RSV F protein. Briefly, lentil lectinis a metalloprotein containing calcium and manganese, which reversiblybinds polysaccharides and glycosylated proteins containing glucose ormannose. The RSV F-containing anion exchange flow through fraction wasloaded onto the lentil lectin affinity chromatography resin (CaptoLentil Lectin, GE Healthcare). The glycosylated RSV F proteinselectively binds to the resin while non-glycosylated proteins and DNAare removed in the column flow through. Weakly bound glycoproteins wereremoved by buffers containing high salt and low molar concentration ofmethyl alpha-D-mannopyranoside (MMP).

In addition, the column washes were also used to detergent exchange theNP-9 detergent with the surfactant polysorbate 80 (PS80). To perform thedetergent exchange, the column was incubated with 0.1% PS80 afterbinding of the RSV F glycoprotein to the lentil lectin column. The RSV Fprotein was eluted from the lentil lectin column with a highconcentration of MMP. After elution, the RSV F protein trimers areassembled into micelle nanoparticles composed of RSV F protein trimersand PS80 contained in a detergent core. After detergent exchange therewas a low pH inactivation step followed by incubation on a sulfatecolumn in the presence of buffer with PS80 at 0.1%.

The eluted material was diluted in a solution containing PS80 adequateto provide a Drug Substance (DS) for hulk storage with a molar ratio ofPS80: RSV F protein of about 50. The adequate composition of the DS wasachieved by combining the RSV F nanoparticles in a solution comprisingphosphate buffer at 22 mM sodium phosphate, 0.03% PS80, and a pH of 6.2.At each step during and after detergent exchange, the antigen to PS80ratio in the composition was maintained at a molar ratio between 35 and60. The molar ratio was calculated using the PS80 concentration and RSVF concentration, as measured by ELISA/A280, and their respectivemolecular weights. The molecular weight of PS80 is 1310 and for RSV is65 kD.

Example 2 Preparation of a Vaccine Composition

To provide nanoparticles for an administered vaccine product, the DrugSubstance was diluted into a Drug Product, with a PS80: RSV proteinmolar ratio of about 50. Drug Substance was thawed, diluted and filledinto glass vials or pre-filled syringes for storage at 2-8° C. prior toadministration. The nanoparticles bound to alum adjuvant. The alumadjuvant was added and mixed to ensure about 95% of the nanoparticlesare bound to the alum is bound, meaning about 0.4 mg per 120 μg dose ofRSV F nanoparticle in a 0.5 mL volume.

Example 3 Characterization of RSV F Glycoproteins in Nanoparticles

We analyzed protein structure in the nanoparticles by various analyticaltechniques. FIG. 3 shows that the highest peak of RSV F protein producedcontains palmitoleic acid (Peak 2A). The second largest peak containspalmitic acid (Peak 2B). Residual peaks were obtained lacking eitherfatty acid (Peak 5) and in soluble form (Peak 1). Analysis on SDS-PAGEgel separated additional variants, including the F1+2 protein, F1, F1A,F1B, and F1C portions as well as F2. See FIG. 4 . Analysis of thepeptide structure was performed using peptide mapping. See FIG. 5 . Toassess the glycan structures on the RSV F glycoproteins, HPLC-FLD wasperformed. The results demonstrated that the major glycan structures arefucosylated.

Example 4 Examination of RSV F Nanoparticles by Electron Microscopy

Nanoparticles as prepared in Example 1 were visualized by electronmicroscope. The results confirmed formation of nanoparticles containingthe RSV F glycoproteins surrounding the detergent core. The precisecomposition of the detergent core remains unclear. FIG. 6 illustratesthe types of nanoparticles obtained. The RSV F proteins maintainedtrimer structure even after the detergent exchange. Several types ofnanoparticles were obtained that vary in the number oftrimers/nanoparticle and in morphology. FIG. 6 shows that multipletrimers can be found around the detergent core. In the highlightedportion seven trimers are shown surrounding the detergent. The mainpanel in FIG. 6 illustrates the range of trimers around the detergentcore that are produced. The cartoon structure of the RSV F proteintrimer in the bottom left panel illustrates the orientation of thetrimers with the bottom portion associated with to the detergent core,facilitated by the fatty acids attached to each RSV F glycoprotein.

Example 5 Particle Characterization of RSV F Nanoparticles

Dynamic light scattering (DLS) was utilized to determine the sizedistribution profile of the nanoparticles by measuring changes in lightscattering patterns of particles in Brownian motion. Nanoparticle sizeswere able to be determined as a linear function of the concentration ofionic detergent versus the concentration of the RSV F nanoparticles (seeFIGS. 7 and 8 ).

Analytical ultracentrifugation (AUC) was used to measure the progressionof the sample concentration versus the axis of rotation profile as aresult of the applied centrifugal field. FIG. 8 reveals thatpredominantly two shapes of nanoparticles emerge based upon theconcentration of nanoparticles present. Nanoparticle types obtainedinclude monomeric and dimeric anisotropic rods, and spherical oligomers.Structure intermediates between these two nanoparticle types form atconcentrations between those that result in anisotropic rods andspherical oligomers. FIG. 9 shows that nanoparticle type can becontrolled by modulating the concentration of the RSV F protein, withhigher concentrations (1 mg/mL) resulting in a predominant population ofspherical oligomers, while lower concentrations (0.22 mg/mL) resultingin predominant populations of monomeric,/dimeric; anisotropic rods.These data illustrate that the detergent amounts and the RSV Fconcentration can be controlled to arrive at nanoparticle have aparticular diameter (z-ave) from 20 nm to 60 nm.

Example 6 Enhanced Stability of Nanoparticles: MolecularCharacterization

The five stressors utilized were thermal stress at 50° C. with timepoints at 48 hours, one week, and two weeks; low pH (3.7 at 25° C.) withtime points at 48 hours, four days, and one week; high pH (10 at 25° C.)with time points at 24 hours, 48 hours, and one week; hydrogen peroxideoxidation at 25° C. with time points at 12 hours, 48 hours, and oneweek; and physical agitation at 25° C. with time points at four hours,24 hours, and one week.

After the various stress treatments differences in the primary structurewere assessed. FIG. 10 shows a comparison of how particular regionssurvived stress relative to a control. The data show that thenanoparticles have excellent stability across the entire protein, andthat only the especially harsh oxidation test using hydrogen peroxidewas able to degrade the protein to any particular extent. However, eventhat treatment did not reduce the structural integrity of antigenic siteII, which is the target of palivizumab. Indeed, even with the harshoxidation the RSV-F protein only deteriorated structurally at positions63-82, 237-258, and 360-364. Accordingly, even after being subjected toharsh stress, the nanoparticles remained substantially intact.

FIG. 11 further quantifies nanoparticle stability regarding antigenicsite II. The data show that in response to each of thermal stress, lowpH, high pH, and agitation that the antigenic site II remained intact tothe extent of 90% in each of the samples.

Example 7 Enhanced Stability of Nanoparticles: Maintained ImmunogenicProperties

The stressed vaccine compositions described in Example 6 were evaluatedfor immunogenicity in a murine model. The vaccines were administered tomice via two intramuscular injections across a range of RSV F dosesconsisting of 0.15, 0.45, 1.3, 4, and 12 μg/mL of RSV F protein. RSV Fcomposition stressed under the following conditions, accompanied by acontrol (thawed from storage at −70° C.), were administered: 50° C. fortwo weeks, the pH 10 at 25° C. for one week, 0.5% hydrogen peroxide at25° C. for one week.

The immune response was evaluated with regard to the presence ofanti-RSV F IgG, PCA titers, and the presence of RSV A neutralizingantibodies. The physical and chemical stressors did, not significantlyaffect, in vivo, the RSV F protein immunogenicity. The stressed samplesinduced similar anti-RSV F IgG antibody titers and comparable functionalPCA and RSV A neutralizing titers to those of the unstressed RSV Fnanoparticle vaccine composition control, (See FIGS. 12A-12D).Collectively, these forced degradation studies indicate that even whenexposed to severe environmental stresses the nanoparticles induce potentimmune responses.

Example 8 Protease Resistance of Nanoparticles

Formation of nanoparticles with improved stability is dependent on theamount of PS80 used to produce the nanoparticle. FIG. 13 illustrates adramatic improvement in stability up to 18 months when nanoparticleswere formed with 0.03% PS80 (i.e. a molar ratio of 55) compared to0.015% (i.e. a molar ratio of 27). The left panel shows an SDS-PAGE ofnanoparticles produced at the two concentrations at time zero. The datashows that similar results to a reference preparation were obtained forboth the nanoparticle preparation. Specifically, both show robustsignals for F1, and F1+2 illustrating essentially no degradation.Aliquots of each preparation were then incubated at 4° C. for 18 monthsand then run again on SDS-PAGE. The data on the right panel as well asthe table illustrates that nanoparticles containing only 0.015% in theparticle resulted in truncated F1. In contrast, nanoparticles preparedwith 0.03% in the particle illustrated excellent resistance to protease.We think that maintaining a correct ratio of detergent and proteinresults in a nanoparticle having an orientation of glycoprotein withprotease-sensitive portions protected, possibly via some sterichindrance mechanism. We further observed that concentrations of PS80 ator above 0.06% increased aggregate formation. Taken together the datashow that optimum PS80 levels are about 0.03% to about 0.05% fornanoparticle stability.

Example 9 Purification of HA Nanoparticles

The TMAE column was pre-equilibrated with Buffer A2 (25 mM Tris pH7.5,70 mM NaCl, 0.02% NP9 for 0.5 CV at Flow Rate: 91.7 cm 30 mL/min. Samplewas loaded at 20 mL/min (25min resident time) and then washed with EQbuffer A1 (25 mM Tris pH7.5. 70 mM NaCl, 0.02% NP-9). The purifiedsample was then eluted using 1.25 CV 15% Buffer B (25 mM Tris pH8, 1MNaCl, 0.02% NP9) followed 1.1 cv 100% B. A representative chromatogramis shown in FIG. 18B. The product from the TMAE column was applied to aLentil lectin affinity chromatography column pre-equilibrated withBuffer A11: 25 mM sodium phosphate pH6.0, 10 mM NaCl, 0.05% PS80 for 3CV (Flow Rate: 147cm/h 13mL/min). Sample was loaded at 9.4 min residenttime−6.5mL/min−73.5cm/h. After loading, high-salt washing was performedwith 3 CV Buffer A12 (25 mM sodium phosphate pH6.0, 500 mM NaCl, 0.5%NP-9). After the first wash, detergent exchange was performed by washingthe column with 6 CV of Buffer All (25 mM sodium phosphate pH6.0, 10 mM,NaCl, 0.05% PS80), Nanoparticles containing PS80 were then eluted with100% B for 3 CV, B1: Buffer B: 25 mM sodium phosphate pH6.0, 10 mM NaCl,0.05% PS80, 500 mM Methyl-alpha-D-Mannopyronoside. A representativechromatogram trace is shown in FIG. 18C. The product from the lentillectin column was applied to a sulfate column with 3 CV Buffer A1 (25 mMsodium phosphate pH6.0, 10 mM NaCl, 0.05% PS80), washed with 2 CV BufferA1 then eluted with 100% Buffer B1 (25 mM sodium phosphate pH 7.5, 500mM NaCl, 0.05% PS80). The eluted product was then combined 1:1 with 50mM sodium phosphate pH 9 and sterile filtered. The final product was atpH 7.2. A chromatogram is shown in FIG. 18D. FIG. 18E provides a gel andwestern blot of various products obtained during the purificationprocess from the TMAE and LL columns. FIG. 18F shows eluate from theS03-column.

Example 10 Analysis of HA Nanoparticle Purity

HA nanoparticles were prepared as outlined in FIGS. 17 and 18 . Wemeasured purity of multiple HA nanoparticle preparations using HAsequences derived from various strains (A/New Hampshire/1/2015,A/Switzerland/9715293/2013, A/Hong Kong/4801/2014, B/Phuket/3073/2013,and B/Brisbane/60/2008). The data showed that high purity preparationswere obtained in all cases. Analysis by gel densitometry showed purityabove 93% and ranging from 93% to 97%. See FIGS. 19A to 19H. We alsoanalyzed purity of the three A subtype strains by RP-HPLC and found thatpurity was 83% to 85%. See FIG. 19I. In addition, we measurednanoparticle size. The nanoparticles showed a diameter between 22.0 nmand 29.9 nm. See FIG. 19 .

Example 11 Analysis of HA Ultrastructure

Electron microscopy was performed to assess the structure of the HAnanoparticles. We found that, like other glycoproteins, the HAglycoproteins formed trimers that were associated with the PS80detergent core. Each detergent core contained multiple trimers. See FIG.20 . Using cryo-EM 2D class averaging we docked the HA trimers ontonanoparticles. FIG. 21A shows results of these in silico dockingexperiments. The upper panels show the fit for an HA trimer onto a firstnanoparticle. The lower panel shows the fit onto another stalk of ananoparticle

For comparison, docking on VLPs was performed. The VLPs contain a lipidbilayer into which the HA protein is anchored. See FIG. 21B. The centerpanels show HA protein structure overlaid onto the stalk emanating fromthe bilayer. The right hand upper and lower panels shows free HA EMmicrograph looking straight down onto the HA trimers, alone (upperpanel) and with the corresponding HA structure overlaid on the EMpicture (lower panel).

Example 12

Immunogenicity Analysis of HA Nanoparticles Co-Adminstered with RSV FNanoparticles

Immunogenicity of nanoparticles in vaccines in a mouse model wasassessed. A combination of nanoparticles containing two antigens(influenza HA protein from A/Switzerland H3 sub-type and RSV F protein)was administered. Each nanoparticle was also administered separately.The vaccines were administered alone or with adjuvants AlPO₄ or Matrix Msaponin adjuvant. FIG. 22 shows the treatments administered to groups1-10. Group 10, the control, was not treated. Treatments wereadministered at. Days 0 and 21. We measured HAI against heterologous andhomologous challenges. See FIGS. 23A and 23B. FIG. 23A shows thatMatrix-adjuvanted. HA nanoparticles stimulated particularly robustresponses against homologous challenge. FIG. 23B shows that a robust HAIresponse was also obtained against heterologous influenza strainA/Texas/50/2012 when administered with Matrix M, and this response wasnot affected by co-administration with the RSV F nanoparticle.

We also measured the ability of the RSV F nanoparticle component toinduce formation of antibodies that compete with Palivizumab. FIG. 23C.The data shows that RSV F nanoparticles administered alone and witheither AlPO4 or Matrix M induced substantial antibody titer of 80 μg/mLto around 700 μg/mL. When both RSV F and influenza nanoparticle wereinduced a lowered response of around 20 μg/mL to 40 μg/mL was obtainedin the absence of adjuvant or with AlPO₄. With Matrix M, however, theRSV F response was robust when administered alone or in combination withthe HA nanoparticle. Measurement of RSV neutralizing antibodies showed asimilar pattern to PCA antibodies. See FIG. 23D.

In addition, to antibody responses, we measured T cell responses inducedby the vaccines against RSV and against influenzaA/Switzerland/9715293/2013. FIGS. 23E and 23F. The data show robustinduction of IFNγ against both targets when Matrix M was used asadjuvant.

Example 13 Trypsin-Resistant Nanoparticle Production

Certain approaches to producing influenza can result in trypsinsensitivity of the HA protein, which alters folding leading to reducedimmunogencity and stability of vaccine formulations. To produce atrypsin-resistant HA nanoparticle we used a detergent exchange approachemploying neutral pH buffers. See FIGS. 24A-C.

Sf9 cells were infected with HA nanoparticle BV vectors MOI=0.1; 3E6cells/ml. Cells were harvested on day 3 and lysed with buffered 0.5%NP9; pH 7.5. We next performed anion exchange chromatography (FractogelTMAE; EMD). FIG. 24B shows an illustrative chromatogram. The flowthrough contains the HA. Following anion exchange column, detergentexchange was performed using a Capto Lentil Lectin column (GE) using ahigh salt/detergent wash containing 0.01% PS80; pH 7.2 and eluted alsoin 0.01% PS80. FIG. 24C shows an illustrative chromatogram for thedetergent exchange process. Finally, we used Tangential flow filtration(TFF): 50 kD MWCO filter; pH 7.2 to produce the bulk drug substance(BDS) used to store the product. At the TFF stage and afterwards PS80was maintained in 0.05%, in a buffer with pH 7.2.

Example 14 Trypsin-Resistant Nanoparticle Analysis

HA nanoparticles from various strains using the process described inExample 13 and production was assessed. FIG. 25A shows that yield ofstrain A/New Hampshire/1/2015 (H1N1) was about 20 mg/L. B straininfluenza nanoparticles also had excellent productivity and purity. FIG.25B shows yield and purity of a B strain, B/Brisbane/60/08 HA, assessedas the bulk drug substance. Yield was about 30 nag/L. FIG. 25C showsyield of an H3N2 strain showing the process gave about 96% purity and ayield of about 20 mg/L.

Thermodynamic Stability Analysis

FIG. 25D provides a thermodynamic profile comparison between HAnanoparticles produced using the trypsin-resistant neutral pH approachin Example 13 versus approaches using low pH purifications steps, suchshown in FIG. 18 . The Differential Scanning calorimetry (DSC) methodwas used to determine the thermodynamic profile of macromolecules insolution, specifically, by measuring the difference in heat energyuptake between a sample solution and appropriate reference(buffer/solvent) by varying the temperature in a controlled manner. Inrelation to Flu HA samples, DSC has allowed us to visualize transitionmidpoints (Tm's), defined as temperatures where half the protein isdenatured/unfolded and half in the native/folded state.

Additionally, DSC has given us information regarding proteinconformation and the estimated stability profiles can be roughlyextrapolated for each HA strain undergoing differing process conditions.The difference between HA purified by a process step that exposed the HAto pH 6.0 during purification vs RA purified via an alternate step (e.g.MMC resin or TFF membrane) is shown using the B/Brisbane HA as anexample. The data shows Tm values are higher in intensity, show a shiftto higher onset of Tm for the main peak and sharper peaks suggestingproper folding of the HA when purified by the alternate step. The datafor the HA exposed to pH 6.0, however shows Tm values that have eithergreatly diminished in intensity, show earlier onset for the Tm of themain peak, have significantly broadened peaks displaying slowasymmetrical unfolding, and/or contain misfolded proteins andaggregation peaks at higher temperatures. Similar profiles were observedfor the other strains (A/Cal, A/Hong Kong, A/New Hampshire). While lowpH gives rise to nanoparticles as discussed above, and while they mayhave certain application, based on these three observations in the DSCdata, we conclude that the alternate process steps/conditions usingneutral pH yield HA protein with significantly better thermodynamic andpotentially improved stability profiles.

Trypsin Resistance

FIG. 26 illustrates the improved trypsin resistance obtained whennanoparticles were produced as described in Example 13. To test trypsinsensitivity, HA samples were diluted to 0.24 mg/mL, incubated withdecreasing trypsin at 37° C. for 60 min, Trypsin inhibitor was added tostop the digestion, and then SDS-PAGE analysis was performed. Comparingthe left panel and right panel of FIG. 26 illustrates the enhancedtrypsin resistance. Purified HA nanoparticles made in Sf9 insect cellsare HA0. When exposed to trypsin HA0 is cleaved to HA1 and HA2 at ArgAA344 in H1. Correctly folded HA trimers will resist further cleavagewhen incubated with increasing concentrations of trypsin. Neutral pHpurified B/Brisbane/60/08 is resistant to trypsin and is correctlyfolded (left panel). Acid pH purified B/Brisbane/60/08 HA1 is trypsinsensitive and misfolded (right panel). FIGS. 26B and 26C illustrate thattrypsin resistance is achieved for a variety of strains. FIG. 26B showsstrain A/Hong Kong/4801/2014. Neutral pH purified A/Hong Kong/4801/2014(H3N2) is resistant to trypsin and thus is correctly folded (leftpanel). Acid pH purified A/Hong Kong/4801/2014 (H3N2) HA1 is trypsinsensitive and not correctly folded (right panel).

Similar data was obtained with A/New Hampshire/1/2015, and H1N1sub-type. Like the other strains, acid-purified H1N1 was misfolded (datanot shown) whereas the neutral pH purified protein was trypsin-resistantand correctly folded.

Comparison with Commercial Flu Vaccines

Previous approaches to producing recombinant influenza vaccines have notmet with widespread success. To investigate whether egg-produced orrecombinantly-produced flu vaccine exhibited trypsin-resistance wecompared trypsin-sensitivity in egg-produced and recombinant vaccines(Fluzone® and Flublok®, respectively) using the same protocols as above.Specifically, undiluted vaccines were incubated with varying amounts oftrypsin at 37° C. for 60 min, then trypsin inhibitor was added, 2×Sample Buffer was added, and heated at 70° C. for 10 min before SDSpage.

We found that the egg-produced variant showed trypsin resistance.Specifically, commercial trivalent egg-derived high dose Fluzonevaccine, which is cleaved to HA1 and HA2 and HA1, is resistant totrypsin digestion. In contrast, commercial trivalent recombinant HAFlublok vaccine when exposed to trypsin is converted to HA1 and HA2 andHA1 polypeptides, and is sensitive to trypsin. FIG. 27 (right panel).These results demonstrate at least one of the strains is denaturedpossibly due to purification at pH 5.89 (See for example, Wang et al.Vaccine. 24 (2006); 2176.).

Thus, recombinant influenza vaccines that are commercially availablesuffer from misfolding that may arise from production under low pHconditions and likely explains, at least in part, their poorimmunogenicity and the lack of widespread adoption to date.

In contrast the methods disclosed herein confirm that purifying HAnanoparticles using buffers of at least pH7.0 reduces or eliminatesmisfolding of the HA protein that occurs when HA proteins are exposed toacid conditions during purification.

Example 15 Construction of Ebola Virus Glycoprotein Nanoparticles

The wild-type fill-length, unmodified EBOV glycoprotein (GP) gene fromthe 2014 Makona Ebola virus was cloned into recombinant baculovirus andexpressed in Spodoptera frugiperda Sf9 insect cells. After expression,the N-terminal signal peptide is cleaved and the mature protein ispurified and formed into nanoparticles. Purified Ebola virus GP(EBOV/Mak GP) nanoparticles are composed of multiple GP trimersassembled into spherical particles 36+4 nm (as measured by dynamic lightscattering). Recombinant GP nanoparticles have a core region whichcontains the glycoprotein 2 (GP2) ‘fusion subunits’ with 2-9 or up to 15“chalice-like” glycoprotein 1 (GP1) trimers ‘attachment subunits’extending outward.

For co-administering Matrix-M, a saponin-based adjuvant consisting oftwo populations of individually formed 40 nm sized. Matrix particles,was used. The Matrix-M used was 85% Matrix-A and 15% Matrix-C. TheMatrix particles were formed by formulating purified saponin fromQuillaja saponaria Molina with cholesterol and phospholipid.

Example 16 Immunization and Protocols

Balb/c mice (6-8 weeks old; Harlan Laboratories Inc., Frederick, Md.)were housed in groups of 10 and immunized by subcutaneous (SC) orintramuscular (IM) administration. Phosphate buffered saline (PBS) wasused as placebo. Blood samples for serum were collected via theretro-orbital route. Prior to blood collection, animals wereanesthetized with isoflurane.

Mice (n=10 per group) were immunized by IM administration (50 μlinjection volume) at Days 0 and 21 with EBOV/Mak GP alone or mixed withAlPO₄ (50 μg) or Matrix-M adjuvant (2.5 μg or 5 μg). Blood samples werecollected at Days 0, 14, 21, 28 and 60. Spleen and bone marrow sampleswere collected at days 28 and 60. Spleen and bone marrow samples weresuspended in PBS containing 2% fetal bovine serum (FBS) for furtherpreparation.

Serum samples from day 28 were evaluated for anti-EBOV/Mak neutralizingantibody responses at U.S. Army Medical Research Institute of infectiousDiseases, Fredrick, Md. using a pseudovirion neutralization reporterassay. A hantavirus pulmonary syndrome (HPS) DNA vaccine delivered usinga spring-powered jet injector elicits a potent neutralizing antibodyresponse in rabbits and nonhuman primates. Curr Gene Ther. 2014; 14:200-210. For this assay, the vesicular stomatitis virus G protein wasremoved and replaced with luciferase reporter. This VSV luciferaseexpressing core was pseudotyped using the plasmid pWRG/EBOV-Z76 (opt)that expresses the Zaire Ebola virus 1976 (Mayinga) GP. The plasmid usedto provide the pseudotyping Ebola GP was pWRG/EBOV/Mak-Z76 (opt)expressing the Zaire Ebola virus 1976 (Mayinga) GP. PsVs were preparedin 293T cells. Mouse sera were heat-inactivated at 56° C. for 30 minutesand then an initial 1:20 dilution was prepared followed by five-foldserial dilutions in Eagle's Minimum Essential Medium (EMEM) (LifeTechnologies) supplemented with 10% (vol/vol) heat inactivated FBS, 100IU/mL penicillin, and 100 μg/mL streptomycin (cEMEM), Ebola GP PsVs werediluted in cEMEM. An equal volume of PsVs solution containing 4×10³focus-forming units and 10% guinea pig complement (Cedarlane) was addedto the sera dilutions for a final starting dilution of 1:40 and thenincubated overnight at 4° C. Vero cell monolayers seeded in clear-bottomblack 96-well plates (Corning) were infected with 50 μl of eachPsVs-serum mixture and then incubated at 37° C. for an additional 18-24hours. The medium was discarded, the cells were lysed, and luciferasesubstrate was added according to the Renilla Luciferase Assay Systemprotocol (Promega #E2820). The flash luciferase signal was measuredusing a Tecan M200 microplate reader, Raw values were exported toGraphPad Prism version 6.04, where the data were baseline-corrected tothe untreated PsVs signal. The data were fit to four parameter logisticnonlinear regression models using GraphPad Prism and then PsVNA 50%(PsVNA50) neutralization titers were interpolated from the curves foreach sample. Each sample was analyzed in triplicate. The assay positivecontrol was serum from a rabbit vaccinated three times with thepWRG/EBOV-Z76 (opt), a Zaire Ebola virus 1976 Mayinga GP DNA vaccine.

EBOV/Mak GP specific serum antibodies were quantitated by enzyme linkedimmunosorbent assay (ELISA). Briefly, NUNC MaxiSorp microtiter plateswere coated with 2 μg/mL of EBOV/Mak GP (Novavax) overnight at 2-8° C.Unreacted surface was blocked with StartingBlock Blocking Buffer(Pierce) for one hour at room temperature (RT). The plates were reactedsequentially at RT with 5-fold serial dilutions of serum samplesstarting from 1:100 (two hours), goat anti-mouse IgG (or IgG land IgG2a)conjugated to horseradish peroxidase (HRP) (Southern Biotech) (onehour), peroxidase substrate 3,3,5,5-Tetramethylbenzidine (FMB) (Sigma)(ten minutes) and TMB Stop Buffer (Scy Tek Laboratories). The plateswere washed three times with PBS/Tween (Quality Biologicals) beforeaddition of the HRP conjugate and TMB reagent.

The plates were read at 450 nm in SpectraMax plus plate readers(Molecular Devices). SoftMax pro software (Molecular Devices) was usedto fit concentration-responses to a 4-parameter fit curve. Antibodytiters were defined as the reciprocal of the highest dilution in whichthere was a 50% maximum antibody binding (EC₅₀) response. If the serumIgG titer is out of the lower detection range, then a titer of <100(starting dilution) was reported and a value of 50 assigned to thesample to calculate the group geometric mean titer (GMT). Mouseanti-EBOV/Mak GP monoclonal antibody (mAb) (4F3) from IBT Bioservices(Gaithersburg, Md.) was used as the positive control.

ELLSPOT Assay, Assessing IFN-γ and EBOV/Mak GP-Specific IgG-SecretingCells

Single cell suspensions were prepared from individual spleens by gentlygrinding the tissues using the plunger of a syringe. Single bone marrowcell suspension was prepared by flushing PBS containing 2% FBS throughthe bone using a syringe with a 21-gauge needle. Cells were washed twicewith PBS containing 2% FBS and counted. IFN-γ ELISPOT assays wereperformed using mouse IFN-γ ELISPOT kits (eBioscience, San Diego,Calif.) according to the manufacturer's procedure. Briefly, anti-IFN-γantibody (15 μg/ml in PBS) was used to coat ELISPOT plates (Millipore,Darmstadt, Germany) at 100 μl/well, overnight at 4° C. The plates werewashed four times with PBS and blocked with RPMI1640 medium plus 5% FBSfor 1-2 hours at room temperature. A total of 3×10⁵ splenocytes in avolume of 200 μl were stimulated with pools of 15-mer EBOV GP peptideswith 11 overlapping amino acids (2.5 μg/ml) covering the entire EBOV GPsequence. Phorbol myristic acetate (PMA) (50 mg/ml) plus ionomycin (200ng/ml) was used as positive control and medium as negative control. Eachstimulation condition was carried out in triplicate. Assay plates wereincubated overnight at 37° C. in a 5% CO₂ incubator and the signals weredeveloped based on manufacturer's instructions. Spots were counted andanalyzed. using an ELISPOT reader and Immunospot software (CellularTechnology, Ltd., Shaker Heights, Ohio). The Ebola-GP specific spotnumber was obtained by subtracting the background number in the mediumcontrols from the GP-peptide stimulated wells. Data shown in the graphare the average of triplicate wells. To measure GP-specificIgG-secreting cells, ELISPOT plates were coated with EBOV/Mak GP (2.5μg/ml in PBS) and incubated overnight at 4° C. Plates were washed andblocked as described above. Triplicates of 3-5×10⁵ splenocytes or bonemarrow cells per well were plated and the plates were incubatedovernight at 37° C. On the second day the plates were washed, and goatanti-mouse IgG-HRP was added and incubated for 1.5 hours. Spots weredeveloped and counted as described above. The average spot number fromtriplicate wells were calculated and presented.

Surface Staining for Cell Phenotypes and Intracellular Staining forCytokines

For surface staining, cells were first incubated with anti-CD16/32antibody (clone 2.4 G2) to block the Fc receptor. To characterize thegerminal center cells, 1×10⁶ of fresh splenocytes were incubated at 4°C. for 30 min with a mixture of the following antibodies: B220-PerCP,CD19-APC, GL7-BV421, CD95-PE-Cy7 (BD Biosciences, CA) and the yellowLIVE/DEAD® dye (Life Technologies, NY). Cells were washed twice andsuspended in PBS containing 2% FBS for analysis. To stain T follicularhelper cells, 1×10′ fresh splenocytes were incubated with CXCR5-Biotin,washed two times, then incubated with a mixture of antibodies includingCD3-BV650, B220-PerCP, CD4-PE-Cy7, Streptavidin-BV421, PD-1-APC,CD69-FITC and CD49b-PE (BD Biosciences, CA) and the yellow LIVE/DEAD®dye (Life Technologies). Cells were washed twice and suspended in PBScontaining 2% FBS for analysis.

For intracellular staining for cytokines, splenocytes were cultured in a96-well U-bottom plate at 1 ×10⁶ cells per well. The peptide stimulationwas performed as described for the ELISPOT cultures. The plate wasincubated 6 hours at 37° C. in the presence of BD GolgiPlug™ and BDGolgiStop™ (BD Biosciences). Cells were washed twice, incubated for 20min at 4° C. with a mixture of antibodies for cell surface markers,including CD3-BV 650, CD4-PerCP, CD8-FITC, CD44-APC-Cy7 and CD62L-PE-Cy7(BD Pharmingen, CA) and the yellow LIVE/DEAD® dye (Life Technologies,NY). After two washes, cells were fixed with Cytofix/Cytoperm (BDBiosciences) for 30 min at 4° C., followed by two washes with BDPerm/Wash™ (BD Biosciences). Cells were incubated with antibodies toIFN-γ -APC, IL-2-BV 421 and TNFα-PE (BD Biosciences) overnight at 4° C.The cells were washed and re-suspended in 1×BD Perm/Wash buffer for dataacquisition. All staining samples were acquired using a LSR-Fortessaflow cytometer (Becton Dickinson, San Jose, Calif.) and the data wereanalysed with Flowjo software version Xv10 (Tree Star Inc., Ashland,Oreg.).

Statistical analysis was performed using SAS software version 9.4,Pairwise comparisons with Tukey's adjustment from ANOVA used group asthe independent variable and log-transformed titer result as thedependent variable to determine significance between groups.

Example 17 EBOV/Mak GP Induced Antibody Response and ProtectiveEfficacy.

The immunogenicity of the EBOV/Mak GP nanoparticle vaccine was evaluatedwith and without adjuvant in a mouse model. Mice were vaccinated on Days0, 14 and 28 by SC injection with 5 μg of EBOV/Mak GP alone or EBOV/MakGP formulated in Matrix-M or AlPO₄ adjuvant. Analysis of sera obtainedon day 28 (14 days post second immunization) indicated that the Matrix-Madjuvanted EBOV/Mak GP induced high levels of antigen-specificantibodies against the Mayinga GP with a geometric mean titer (GMT) of26,991. The response obtained following immunization with EBOV/Mak GPwith Matrix-M was significantly higher than those induced by EBOV/Mak GPalone (GMT=266, p=0.001) or EBOV/Mak GP adjuvanted with AlPO₄ (GMT=436,p=0.0001) (FIG. 28A). The AlPO₄ adjuvant offered only marginal increasein anti-EBOV/Mak GP IgG in comparison to the EBOV/Mak GP alone.

The neutralization activity of day 28 sera was analysed using Ebolapseudovirions (PsVs) (FIG. 28B). In the absence of adjuvant,neutralization GMT titer in sera from mice immunized with the EBOV/MakGP alone were 197 and when EBOV/Mak GP was adjuvanted with AlPO₄, lowertiters were observed (GMT=49, p=0.1). Neutralization titers observed insera from mice immunized with EBOV/Mak GP with Matrix-M had a GMT of6,463, thirty-two-fold higher than that obtained with EBOV/Mak GP alone.In this assay, PsVs expressing the EBOV 1976 Mayinga strain GP were usedas PsVs expressing the EBOV/Mak 2014 strain GP were not available. Thus,the assay is measuring the cross-neutralizing activity of anti-EBOV/MakGP against the Mayinga GP.

On day 42, two weeks after the third vaccination given on day 28, micewere challenged by an intraperitoneal inoculation of 1,000 pfu mouseadapted Zaire Ebola virus strain 1976 Mayinga. Control mice started tosuccumb to infection after three days while mice vaccinated withEBOV/Mak GP alone or EBOB/Mak GP adjuvanted with AlPO₄ succumbed at dayfive or six, respectively. Twenty-one days after challenge infection,all mice vaccinated with Matrix-M adjuvanted EBOV/Mak GP and one mousevaccinated with EBOV/Mak GP alone was alive and healthy. In contrast,all other mice were dead or had been euthanized by day 8 due to Ebolavirus infection (FIG. 28C).

Example 18 Kinetics of Ebola-GP IgG, IgG1 and IgG2a Responses

In order to further characterize the immune responses toMatrix-M-adjuvanted EBOV/Mak GP in more detail, two groups of Balb/cmice (10/group) were injected with 5 μg EBOV/Mak GP adjuvanted witheither 2.5 or 5 μg of Matrix-M. Groups of mice injected with PBS,EBOV/Mak GP alone or EBOV/Mak GP with AlPO₄ served as controls. At days14, 21, 28 and 60, EBOV/Mak GP-specific IgG and IgG subclasses (IgG1 andIgG2a) were measured by ELISA.

At day 14 following the first injection, all mice injected with EBOV/MakGP with Matrix-M (2.5 or 5 μg) responded with EBOV/Mak GP-specific IgG(GMT=755 and 1,499 respectively, data not shown). None of the 10 mice inthe EBOV/Mak GP group and EBOV/Mak GP with AlPO₄ group generatedEBOV/Mak GP specific IgG (data not shown). By day 21, the IgG responseto EBOV/Mak GP increased further in the Matrix-M-adjuvanted groups (FIG.29A). There was still no response in the groups given EBOV/Mak GP aloneor with EBOV/Mak GP with AlPO₄. All mice received a second injection atday 21. At day 28 there was a robust increase in IgG responses in themice receiving Matrix-M (2.5 or 5 μg) with ELISA GMT titers of 3.0×10⁵and 4.9×10⁵ respectively (FIG. 29A). At days 28 and 60 in the EBOV/MakGP alone and with AlPO₄ groups, specific IgG responses were detected insome of the mice, but were significantly lower than in the miceimmunized with EBOV/Mak GP with Matrix-M (FIG. 29A). By day 60, theanti-GP IgG titers induced by EBOV/Mak GP with 2.5 or 5 μg Matrix-M werenot significantly reduced compared to day 28 and were 67-fold and139-fold higher, respectively, than in the EBOV/Mak GP with AlPO₄ groups(FIG. 29A).

The EBOV/Mak GP-specific IgG1 and IgG2a responses were also determined.Similar to total IgG, Matrix-M adjuvanted EBOV/Mak GP vaccine inducedhigh anti-GP IgG1 and IgG2a levels at days 28 and 60 (FIGS. 29B and29C). In contrast, only one out of 10 mice given EBOV/Mak GP alone andfour of 10 mice given EBOV/Mak GP with AlPO₄ produced low levels of IgG1at day 28 (FIG. 29B). At day 60, antigen-specific IgG1 was detected inserum from all five remaining mice in the group given EBOV/Mak GP withAlPO₄, but the average titer was 51- and 41-fold lower than in thegroups given EBOV/Mak GP with 2.5 or 5.0 μg Matrix-M (FIG. 29B),respectively. Furthermore, EBOV/Mak GP alone did not induce detectableIgG2a antibody at days 28 and 60.

Example 19 CD4+, CD8+, and Multifunctional T Cell Response

We next assessed the T cell response to the different EBOV/Mak GPformulations by measuring the number of IFN-γ secreting T cells after exvivo stimulation of spleen cells with EBOV/Mak GP peptides in an ELISPOTassay. At day 28, IFN-γ secreting cells increased in a Matrix-Mdose-dependent manner in spleens from mice immunized with EBOV/Mak GPwith Matrix-M (FIG. 30A, 30B). The average number of IFN-γ-secretingcells in groups receiving EBOV/Mak GP with 5.0 and 2.5 μg of Matrix-Mwere 17- and 10-fold higher respectively than in the group receivingEBOV/Mak GP alone and 8- and 5-fold higher respectively than in thegroup receiving EBOV/Mak GP with AlPO₄ (FIG. 30A).

By day 60, the number of IFN-γ secreting cells in spleens from miceimmunized with EBOV/Mak GP with 5 μg of Matrix-M was still 12-foldhigher than in spleens from mice immunized with EBOV/Mak GP alone and3-fold higher than in spleens from mice immunized with EBOV/Mak GP withAlPO₄ (FIG. 30B). The increased numbers of IFN-γ secreting cells inspleens from mice immunized with EBOV/Mak GP with 2.5 μg Matrix-M werealso maintained at day 60 but at a lower level than with 5 μg Matrix-M.

We further assessed Matrix-M induced CD4+ and CD8+ T cell responses byintracellular staining of cytokines combined with cell surface markers.Analysis of splenocytes by flowcytometric staining at day 28 showed thatboth CD4+ and CD8+ T cells from EBOV/Mak GP with Matrix-M groupssecreted IFN-γ, TNFα and IL-2 (FIGS. 30C and 30D). The frequency ofcytokine-secreting CD4+ and CD8+ T cells was much higher in spleens fromthe EBOV/Mak GP with Matrix-M groups than the baseline or minimalresponses observed in control mice, mice receiving EBOV/Mak GP alone orEBOV/Mak GP with AlPO₄ (FIGS. 30C and 30D). The frequency of T cellsthat simultaneously produce two or more cytokines (IFN-γ, and TNFα andIL-2) was also evaluated at day 28. Both CD4+ and CD8+ T cells producingeither two or three cytokines were detected at marked levels only inspleens from the mice immunized with EBOV/Mak GP with Matrix-M.

Example 20 Germinal Center and T Follicular Helper Cell Responses

The frequency and absolute number of GC B cells in the spleen wereanalysed by flowcytometric staining (FIG. 31A). The analysis showed thatat day 28 (seven days after the 2^(nd) injection of vaccine), EBOV/MakGP adjuvanted with 2.5 and 5 μg of Matrix-M induced responses with a GCfrequency of 1.22 and 2.12% respectively in comparison to placebo,EBOV/Mak GP alone or EBOV/Mak GP with AlPO₄ (0.38, 0.41 and 0.44%respectively) (FIG. 31B). Accordingly, the absolute GC cell number inthe spleen also increased in the groups receiving Matrix-M (FIG. 31C).By day 60, the frequency and absolute number returned to backgroundlevel (FIGS. 31D and 31E)

Analysis of T_(FH) cells frequencies at day 28 showed that EBOV/Mak GPwith 2.5 or 5 μg of Matrix-M induced higher frequencies of T_(FH) cellsthan the EBOV/Mak GP alone or with AlPO₄ (FIGS. 32A and 32B).Accordingly, the absolute number of T_(FH) cells was also enhanced byEBOV/Mak GP with Matrix-M compared to EBOV/Mak GP alone or with AlPO₄(FIG. 32C). By day 60, the frequency and absolute number of T_(FH) cellsretracted to near background levels (FIGS. 32D and 32E).

Example 21 EBOV/Mak GP-Specific Plasma Cells

In order to assess the influence of Matrix-M on the EBOV/Mak GP-specificplasma cells, the number of IgG-producing cells in spleen and bonemarrow was analysed at day 60 after immunization. The analysis at day6.0 demonstrated only a few EBOV/Mak GP-specific IgG-secreting cells(<6/10⁶ splenocytes) in the spleens from mice immunized with Matrix-Madjuvanted EBOV/Mak GP vaccine (FIG. 33A). No IgG-secreting cells weredetected in the spleens from mice immunized with EBOV/Mak GP alone andEBOV/Mak GP with AlPO₄ (FIG. 33A). In contrast, high numbers of EBOV/MakGP-specific IgG-secreting, cells appeared in bone marrow from mice thatreceived Matrix-M adjuvanted EBOV/Mak GP (FIG. 33B), demonstrating theformation of long-lived plasma B cells.

Example 22 Characterization of Antibody Binding to the Nanoparticles

We tested the ability of several anti-Ebola antibodies to bind to thenanoparticles. The antibodies are 13C6, 13F6, 6D8 and KZ52. The EC50curve and values are shown in FIG. 36 and additional binding kineticdata are shown in FIG. 37 . FIG. 38 shows potency data using the 13C6 asa reference. Three of four antibodies exhibited excellent binding to theGP.

Example 23

Non-Human Primate study: Baboon

To confirm the results obtained in mice in a non-human primate model, ababoon study was performed. The study was designed as shown in FIG. 39 .Four groups were formed. Group 1 was the control, Group 2 receivedantigen with AlPO₄. Groups 2 and 3 received antigen, 60 μg and 5 μg,respectively with Matrix-M at 50 μg. Baboons were immunized at 0 and 21days. Robust responses were obtained against both Makona GP and MayingaGP. See FIG. 40 . Additional analysis confirmed that the response waslong-lasting. FIG. 41 shows EC50 values for IgG against Makona at latertimepoints. The data establish the response is sustained.

Additional studies confirm that IFN-γ levels increase substantiallyfollowing immunization. FIG. 42 shows that Matrix M in combination withGP increased more so than with an alum adjuvant. Interestingly, thelower dose of GP, 5 μg, gave a more pronounced increase in IFN-γ levels.TNF-α and IFN-γ responses in T-cells are shown in FIG. 43 with cytokinesresponses shown in FIG. 44 . Again, the response is more pronounced withGP and Matrix M in each case than with alum. These data underscore therobust immune response of the disclosed formulations in the baboonmodel.

Example 24 Non-Human Primate Study: Macaque Study 1

To further confirm the protective effect of the nanoparticles, amacaques study was performed as indicated in FIG. 45 . Macaques wereimmunized intramuscularly at days 0 and 21 with vaccine as shown andchallenged at Day 42. Anti-GP responses were measure at Day 0 and 28. AsFIG. 46 shows, immunized macques showed a dramatic induction of anti-IgGantibodies. The immune response was characterized as shown in FIG. 47 .IFN-γ secreting cells in response to various peptides pools was measuredat weeks 0, 3, and 5. The results demonstrate that immunized macaquesinduced IFN-γ-secreting cells in immunized macaques.

Animal survival was remarkable. FIG. 48 . By day 7, the Ebola viral loadin placebo-treated macaques was 10⁷. By Day 9, the placebo animal waseuthanized. In contrast, 100% of treated animals survived. Remarkably,the immune response was able to render the viral load undetectable byRT-PCR in almost all animals at almost all time points. Animal 33362showed viral load at day 7 that was about 10% above the detectablelimit. By Day 10, however, levels had dropped beneath the ability of theassay to detect them.

Example 25 Non-Human Primate Study: Macaque Study 2

A second study was performed in Macaques. Animals were dosed with 5 μgGP+50 μg Matrix-M at zero weeks, with a follow-on boost at either 3weeks or 6 weeks. Immunized animals were then challenged with wild-typeEbola virus at 9 weeks and 12 weeks, respectively. FIG. 49 .

ELISA data for IgG are shown in FIG. 50 . The left panel shows that 3weeks after the first injection, high titers had developed and weresustained. The right panel illustrates results in animals with a 6-weekgap between administrations. Those animals showed a substantial increasetwo weeks after the second, booster administration, illustrating thebeneficial effect of a prime-boost approach.

The vaccine composition was fully protective in macaques. 18 days afterchallenge with live virus, the saline control-treated mice were alldead. In contrast, 100% of macaques immunized with the vaccinecompositions survived challenge. Collectively, these data confirm thatthe immune responses stimulated by the compositions are protectivewhether a boost administration occurs within 3 weeks or within 6 weeks.

1-24. (canceled)
 25. A nanoparticle comprising: an Ebola virusglycoprotein (GP) and a non-ionic detergent core; wherein the GPcomprises a GP1 domain and a GP2 domain: wherein the GP1 domain and GP2domain are connected via a disulfide bond; wherein the GP2 polypeptideis associated with the non-ionic detergent core and the GP1 polypeptideextends outward; and wherein the GP is a trimer.
 26. The nanoparticle ofclaim 25, wherein the GP comprises the polypeptide sequence of SEQ IDNO:
 29. 27. The nanoparticle of claim 25, wherein the GP is isolatedfrom a Makona, Sudan, Zaire, or Reston Ebola virus strain.
 28. Thenanoparticle of claim 25, herein the GP is at least 80 identical to SEQID NO:79.
 29. The nanoparticle of claim 25, wherein the non-ionicdetergent s selected from the group consisting of PS20, PS40, PS60,PS65, and PS80.
 30. The nanoparticle of claim 25, wherein the non-ionicdetergent is PS80.
 31. The nanoparticle of claim 25, wherein the GP isexpressed in an insect cell.
 32. The nanoparticle of claim 31, whereinthe insect cell is a Spodoptera frugiperda Sf9 cell.
 33. An immunogeniccomposition, comprising: (i) the nanoparticle of claim 25; (ii) asaponin adjuvant; and (iii) a pharmaceutically acceptable buffer. 34.The immunogenic composition of claim 33, wherein the GP of thenanoparticle comprises the polypeptide sequence of SEQ ID NO:
 29. 35.The immunogenic composition of claim 33, wherein the GP of thenanoparticle is isolated from a Makona, Sudan, Zaire, or Reston Ebolavirus strain.
 36. The immunogenic composition of claim 33, wherein theGP of the nanoparticle is at least 80% identical to SEQ ID NO:29. 37.The immunogenic composition of claim 33, wherein the non-ionic detergentof the nanoparticle is selected from the group consisting of PS20, PS40,PS60, PS65, and PS80.
 38. The immunogenic composition of claim 33,wherein the non-ionic detergent of the nanoparticle is PS80.
 39. Theimmunogenic composition of claim 33, wherein the GP of the nanoparticleis expressed in an insect cell.
 40. The immunogenic composition of claim39, wherein the insect cell is a Spodoptera frugiperda Sf9 cell.
 41. Theimmunogenic composition of claim 33, wherein the saponin adjuvantcomprises at least two iscom particles; wherein the first iscom particlecomprises fraction A of Quillaja saponaria Molina and not fraction C ofQuillaja saponaria Molina; and wherein the second iscom particlecomprises fraction C of Quillaja saponaria Molina and not fraction A ofQuillaja saponaria Molina.
 42. The immunogenic composition of claim 41,wherein fraction A of Quillaja saponaria Molina accounts for 50-99% byweight and fraction C of Quillaja saponaria Molina accounts for theremainder, respectively, of the sum of the weights of fraction A ofQuillaja saponaria Molina and fraction C of Quillaja saponaria Molina inthe adjuvant.
 43. The immunogenic composition of claim 41, whereinfraction A of Quillaja saponaria Molina and fraction C of Quillajasaponaria Molina account for about 85% by weight and about 15% byweight, respectively, of the sum of the weights of fraction A ofQuillaja saponaria Molina and fraction C of Quillaja saponaria Molina inthe adjuvant.
 44. A method of stimulating an immune response againstEbola virus in a subject comprising administering the immunogeniccomposition of claim
 33. 45. The method of claim 44, wherein the GP ofthe nanoparticle of the immunogenic composition comprises thepolypeptide sequence of SEQ ID NO:
 29. 46. The method of claim 44,wherein the GP of the nanoparticle of the immunogenic composition isisolated from a Makona, Sudan, Zaire, or Reston Ebola virus strain. 47.The method of claim 44, wherein the GP of the nanoparticle of theimmunogenic composition is at least 80% identical to SEQ ID NO:29. 48.The method of claim 44, wherein the nonionic detergent of thenanoparticle of the immunogenic composition is selected from the groupconsisting of PS20, PS40, PS60, PS65, and PS80.
 49. The method of claim44, wherein the nonionic detergent of the nanoparticle of theimmunogenic composition is PS80.
 50. The method of claim 44, wherein theGP of the nanoparticle of the immunogenic composition is expressed in aninsect cell.
 51. The method of claim 50, wherein the insect cell is aSpodoptera frugiperda Sf9 cell.
 52. The method of claim 44, wherein thesaponin adjuvant of the immunogenic composition comprises at least twoiscom particles; wherein the first iscom particle comprises fraction Aof Quillaja saponaria Molina and not fraction C of Quillaja saponariaMolina; and wherein the second iscom particle comprises fraction C ofQuillaja saponaria Molina and not fraction A of Quillaja saponariaMolina.
 53. The method of claim 52, wherein fraction A of Quillajasaponaria Molina accounts for 50-99©r by weight and fraction C ofQuillaja saponaria Molina accounts for the remainder, respectively, ofthe sum of the weights of fraction A of Quillaja saponaria Molina andfraction C of Quillaja saponaria Molina in the adjuvant.
 54. The methodof claim 52, wherein fraction A of Quillaja saponaria Molina andfraction C of Quillaja saponaria Molina account for about 85% by weightand about 15% by weight, respectively, of the sum of the weights offraction A of Quillaja saponaria Molina and fraction C of Quillajasaponaria Molina in the adjuvant.